Oligonucleotide inhibition of protein kinase C

ABSTRACT

Compositions and methods are provided for the treatment and diagnosis of diseases associated with protein kinase C. Oligonucleotides are provided which are targeted to nucleic acids encoding PKC. In preferred embodiments, the oligonucleotides contain one or more chemical modifications. Methods of modulating PKC expression and of treating animals suffering from disease amenable to therapeutic intervention by modulating protein kinase C expression using oligonucleotides targeted to PKC are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent application Ser. No. 08/089,996, filed Jul. 9, 1993, issued on Dec. 30, 1997 as U.S. Pat. No. 5,703,054, which in turn is a continuation-in-part of U.S. patent application Ser. No. 07/852,852 filed Mar. 16, 1992, now abandoned.

FIELD OF THE INVENTION

This invention relates to therapies, diagnostics, and research reagents for disease states which respond to modulation of the expression of protein kinase C. In particular, this invention relates to antisense oligonucleotides specifically hybridizable with nucleic acids relating to protein kinase C. These oligonucleotides have been found to modulate the expression of protein kinase C.

BACKGROUND OF THE INVENTION

The phosphorylation of proteins plays a key role in the transduction of extracellular signals into the cell. The enzymes, called kinases, which effect such phosphorylations are targets for the action of growth factors, hormones, and other agents involved in cellular metabolism, proliferation and differentiation. One of the major signal transduction pathways involves the enzyme protein kinase C (PKC), which is known to have a critical influence on cell proliferation and differentiation. PKC is activated by diacylglycerols (DAGs), which are metabolites released in signal transduction.

Interest in PKC was stimulated by the finding that PKC is the major, and perhaps only, cellular receptor through which a class of tumor-promoting agents called phorbol esters exert their pleiotropic effects on cells Gescher et al., Anti-Cancer Drug Design 4:93-105 (1989)!. Phorbols capable of tumor production can mimic the effect of DAG in activating PKC, suggesting that these tumor promoters act through PKC and that activation of this enzyme is at least partially responsible for the resulting tumorigenesis Parker et al., Science 233:853-866 (1986)!.

Experimental evidence indicates that PKC plays a role in growth control in colon cancer. It is believed that specific bacteria in the intestinal tract convert lipids to DAG, thus activating PKC and altering cell proliferation. This may explain the correlation between high dietary fat and colon cancer Weinstein, Cancer Res. (Suppl.) 51:5080s-5085s (1991)!. It has also been demonstrated that a greater proportion of the PKC in the colonic mucosa of patients with colorectal cancer is in an activated state compared to that of patients without cancer Sakanoue et al., Int. J. Cancer 48:803-806 (1991)!.

Increased tumorigenicity is also correlated with overexpression of PKC in cultured cells inoculated into nude mice. A mutant form of PKC induces highly malignant tumor cells with increased metastatic potential. Sphingosine and related inhibitors of PKC activity have been shown to inhibit tumor cell growth and radiation-induced transformation in vivo Endo et al., Cancer Research 51:1613-1618 (1991); Borek et al., Proc. Natl. Acad. Sci. 88:1953-1957 (1991)!. A number of experimental or clinically useful anti-cancer drugs show modulatory effects on PKC. Therefore, inhibitors of PKC may be important cancer-preventive or therapeutic agents. PKC has been suggested as a plausible target for more rational design of conventional anti-cancer drugs Gescher, A. and Dale, I. L., Anti-Cancer Drug Design, 4:93-105 (1989)!.

Experiments also indicate that PKC plays an important role in the pathophysiology of hyperproliferative skin disorders such as psoriasis and skin cancer. Psoriasis is characterized by inflammation, hyperproliferation of the epidermis and decreased differentiation of cells. Various studies indicate a role for PKC in causing these symptoms. PKC stimulation in cultured keratinocytes can be shown to cause hyperproliferation. Inflammation can be induced by phorbol esters and is regulated by PKC. DAG is implicated in the involvement of PKC in dermatological diseases, and is formed to an increased extent in psoriatic lesions.

Inhibitors of PKC have been shown to have both antiproliferative and antiinflammatory effects in vitro. Some antipsoriasis drugs, such as cyclosporine A and anthralin, have been shown to inhibit PKC. Inhibition of PKC has been suggested as a therapeutic approach to the treatment of psoriasis Hegemann, L. and G. Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., p. 357-368, CRC Press, Boca Raton, Fla., 1992!.

The oligonucleotides of the invention are useful in the therapeutic treatment of diseases associated with PKC. Such diseases include hyperproliferative and inflammatory conditions including psoriasis, tumors and cancers, for example glioblastoma, bladder cancer, breast cancer, lung cancer and colon cancer.

PKC is not a single enzyme, but a family of enzymes. At the present time at least seven isoforms (isozymes) of PKC have been identified: isoforms α, β, and γ have been purified to homogeneity, and isoforms δ, ε, ζ and η have been identified by molecular cloning. These isozymes have distinct patterns of tissue and organ localization (see Nishizuka, Nature, 334:661-665 (1988) for review) and may serve different physiological functions. For example, PKC-γ seems to be expressed only in the central nervous system. PKC-α and -β are expressed in most tissues, but have different patterns of expression in different cell types. For example, both PKC-α and PKC-β are expressed in, and have been purified from, human epidermis. While PKC-α has been detected mainly in keratinocytes of the basal layers of the epidermis, PKC-β is found mainly in the middle layers of the epidermis and Langerhans cells. PKC-η has been found predominantly in the skin and lungs, with levels of expression much higher in these tissues than in the brain. This is in contrast to other members of the PKC family which tend to be most abundantly expressed in the brain Osada et al., J. Biol. Chem. 265:22434-22440 (1990)!. While the PKC isozymes listed here are preferred for targeting by the present invention, other isozymes of PKC are also comprehended by the present invention.

It is presently believed that different PKC isozymes may be involved in various disease processes depending on the organ or tissue in which they are expressed. For example, in psoriatic lesions there is an alteration in the ratio between PKC-α and PKC-β, with preferential loss of PKC-β compared to normal skin Hegemann, L. and G. Mahrle, Pharmacology of the Skin, H. Mukhtar, ed., p. 357-368, CRC Press, Boca Raton, Fla., 1992!.

Although numerous compounds have been identified as PKC inhibitors (see Hidaka and Hagiwara, Trends in Pharm. Sci. 8:162-164 (1987) for review), few have been found which inhibit PKC specifically. While the quinoline sulfonamide derivatives such as 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) inhibit PKC at micromolar concentrations, they exhibit similar enzyme inhibition kinetics for PKC and the CAMP-dependent and cGMP-dependent protein kinases. Staurosporine, an alkaloid product of Streptomyces sp., and its analogs, are the most potent in vitro inhibitors of PKC identified to date. However, they exhibit only limited selectivity among different protein kinases Gescher, Anti-Cancer Drug Design 4:93-105 (1989)!. Certain ceramides and sphingosine derivatives have been shown to have PKC inhibitory activity and to have promise for therapeutic uses, however, there remains a long-felt need for specific inhibitors of the enzymes.

There is also a desire to inhibit specific PKC isozymes, both as a research tool and in diagnosis and treatment of diseases which may be associated with particular isozymes. Godson et al. J. Biol. Chem. 268:11946-11950 (1993)! disclosed use of stable transfection of antisense PKC-α cDNA in cytomegalovirus promotor-based expression vectors to specifically decrease expression of PKC-α protein by approximately 70%. It was demonstrated that this inhibition caused a loss of phospholipase A₂ -mediated arachidonic acid release in response to the phorbol ester PMA. Attempts by the same researchers at inhibiting PKC activity with oligodeoxynucleotides were ultimately unsuccessful due to degradation of oligonucleotides. Ahmad et al. disclose that transfection of the human glioblastoma cell line, U-87, with vectors expressing antisense RNA to PKCα inhibits growth of the glioblastoma cells in vitro and in vivo. Ahmad et al., 1994, Neurosurg. 35:904-908. Diaz-Meco Conde et al. disclose a peptide corresponding to the pseudo-substrate region of PKC-ζ and oligonucleotides antisense to this isozyme. WO Application 93/20101. Alvaro et al. have identified a novel mutant form of PKC associated with tumors and disclose oligonucleotide sequences complementary to the mutant form. WO Application 94/29455.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1(a) and 1(b) are graphical depictions of the effects on PKC expression of antisense oligonucleotides hybridizable with PKC-α. Oligonucleotides are arranged by PKC target region, 5' to 3'.

FIG. 2 is a line graph showing dose-dependent reduction of PKC-α protein levels after oligonucleotide treatment of A549 cells. ▾=ISIS 4632; ▪=ISIS 4649; =ISIS 4636; ▴=ISIS 4648.

FIG. 3 is a bar graph showing reduction of PKC-α mRNA after treatment of A549 cells with oligonucleotides. Hatched bars represent the 8.5 kb transcript, plain bars represent the 4.0 kb transcript.

FIG. 4 is a line graph showing the relationship between deoxy gap length and activity of chimeric oligonucleotides against PKC.

FIG. 5 is a line graph showing dose response curves for chimeric oligonucleotides (all SEQ ID NO: 3) with different deoxy gap lengths.

FIG. 6 is a bar graph showing the effects of several 2'-O-methyl chimeric oligonucleotides of SEQ ID NO: 3 on PKC-α mRNA levels. Hatched bars represent the 8.5 kb transcript, plain bars represent the 4.0 kb transcript.

FIG. 7 is a bar graph and diagram showing the effects of several 2'-O-methyl and 2'-O-propyl chimeric oligonucleotides (6996, 7273) of SEQ ID NO: 3 on PKC-α mRNA levels. Hatched bars represent the 8.5 kb transcript, plain bars represent the 4.0 kb transcript.

FIG. 8 is a bar graph and diagram showing the effects of additional 2'-O-methyl and 2'-O-propyl chimeric oligonucleotides (7008, 7294) of SEQ ID NO: 3 on PKC-α mRNA levels. Hatched bars represent the 8.5 kb transcript, plain bars represent the 4.0 kb transcript.

FIG. 9 is a set of bar graphs showing the effect of additional oligonucleotides on PKC-α mRNA levels. FIG. 9A shows oligonucleotides 6632, 6653 and 6665. FIG. 9B shows oligonucleotides 3521 (for comparison), 7082, 7083 and 7084. Hatched bars represent the 8.5 kb transcript, plain bars represent the 4.0 kb transcript.

FIG. 10 is a line graph showing anti-tumor activity of ISIS 3521. Each dashed line represents tumor volume in one animal treated with control oligonucleotide; each solid line represents tumor volume in one animal treated with ISIS 3521.

FIG. 11 is a set of line graphs showing effect of oligonucleotides on growth of human MDA-MB231 tumors in nude mice. FIG. 11A shows results obtained with ISIS 3521; FIG. 11B shows results obtained with ISIS3527. Each line represents tumor volume in one animal. =control; ∘=oligonucleotide at 60 mg/kg; Δ=oligonucleotide at 6 mg/kg.

FIG. 12 is a bar graph showing effect of 20-mer phosphorothioate oligonucleotides on PKC-π expression in A549 cells.

FIG. 13 is a line graph showing the effect of ISIS 8459 (2' fluoro gapped version of ISIS 3521) on the growth of A549 xenografts in nude mice. All doses (0.006, 0.06, 0.6. 6.0 mg/kg) of ISIS 8459 inhibit tumor growth.

FIG. 14 is a line graph showing a "crossover" experiment to evaluate the effect of ISIS 3521 on U-87 glioblastoma cells in nude mice. The experiment was carried out with oligonucleotide doses of 2 mg/kg and 20 mg/kg and then treatment was switched (arrow). The group which had originally received ISIS 3521 at 20 mg/kg ("high dose-to-control" group, closed triangles) then received saline and the group which had originally received ISIS 3521 at 2 mg/kg ("low dose-to-high dose", open triangles) then received ISIS 3521 at 20 mg/kg. S=sense oligonucleotide (control); AS=antisense oligonucleotide (ISIS 3521) targeted to PKCα.

SUMMARY OF THE INVENTION

In accordance with the present invention, oligonucleotides are provided that are specifically hybridizable with a nucleic acid that encodes PKC. This relationship is commonly denominated as "antisense". In a preferred embodiment, oligonucleotides are provided that are specifically hybridizable with a nucleic acid encoding a particular PKC isozyme or a particular set of PKC isozymes. Such oligonucleotides may be conveniently and desirably presented in a pharmaceutically acceptable carrier and may be combined with another chemotherapeutic agent.

In accordance with preferred embodiments, the oligonucleotides comprise one or more chemical modifications which convey some desired characteristic such as improved target affinity, cellular uptake or stability in the presence of cellular nucleases. Examples of modifications having such utility are 2'-O-alkyl and 2'-fluoro sugar modifications and phosphorothioate backbone modifications.

Also provided are methods for modulating the expression of PKC using the oligonucleotides of the invention. Such methods comprise contacting cells or tissues suspected of containing said gene with oligonucleotides in accordance with the invention. In the context of this invention, to "contact" tissues or cells with an oligonucleotide or oligonucleotides means to add the oligonucleotide(s), usually in a liquid carrier, to a cell suspension or tissue sample, either in vitro or ex vivo, or to administer the oligonucleotide(s) to cells or tissues within an animal.

Other aspects of the invention are directed to methods for diagnostics and therapeutics of animals suspected of having a disease associated with PKC or one of its isozymes.

DETAILED DESCRIPTION OF THE INVENTION

Oligonucleotides have been employed as therapeutic moieties for the treatment of disease states in animals and man. For example, workers in the field have now identified antisense, triplex and other oligonucleotide compositions which are capable of modulating expression of genes implicated in viral, fungal and metabolic diseases.

As examples, U.S. Pat. No. 5,135,917, issued Aug. 4, 1992, provides antisense oligonucleotides that inhibit human interleukin-1 receptor expression. U.S. Pat. No. 5,098,890, issued Mar. 24, 1992 in the name of Gewirtz et al., is directed to antisense oligonucleotides complementary to the c-myb oncogene and antisense oligonucleotide therapies for certain cancerous conditions. U.S. Pat. No. 5,087,617, issued Feb. 11, 1992, provides methods for treating cancer patients with antisense oligonucleotides. U.S. Pat. No. 5,166,195 issued Nov. 24, 1992, provides oligonucleotide inhibitors of HIV. U.S. Pat. No. 5,004,810, issued Apr. 2, 1991, provides oligomers capable of hybridizing to herpes simplex virus Vmw65 mRNA and inhibiting replication. U.S. Pat. No. 5,194,428, issued Mar. 16, 1993, provides antisense oligonucleotides having antiviral activity against influenzavirus. U.S. Pat. No. 4,806,463, issued Feb. 21, 1989, provides antisense oligonucleotides and methods using them to inhibit HTLV-III replication. U.S. Pat. No. 5,286,717 (Cohen et al.), issued Feb. 15, 1994, is directed to a mixed linkage oligonucleotide phosphorothioates complementary to an oncogene; U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423 (Cohen et al.) are directed to phosphorothioate oligonucleotide analogs used to prevent replication of foreign nucleic acids in cells. Antisense oligonucleotides have been safely administered to humans and clinical trials of several antisense oligonucleotide drugs, targeted both to viral and cellular gene products, are presently underway. The phosphorothioate oligonucleotide, ISIS 2922, has been shown to be effective against cytomegalovirus retinitis in AIDS patients. BioWorld Today, Apr. 29, 1994, p. 3. It is thus established that oligonucleotides can be useful therapeutic instrumentalities and can be configured to be useful in treatment regimes for treatment of cells and animal subjects, especially humans.

Current agents which modulate the activity or metabolism of protein kinase C exhibit many unacceptable side effects due to their lack of specificity, or they exhibit only limited effectiveness in inhibiting the enzyme. The instant invention circumvents problems encountered by prior workers by modulating the production of the enzyme, rather than inhibiting the enzyme directly, to achieve the therapeutic effect. In the instant invention, the oligonucleotide is designed to bind directly to mRNA or to a gene, ultimately modulating the amount of PKC protein made from the gene.

This relationship between an oligonucleotide and its complementary nucleic acid target to which it hybridizes is commonly referred to as "antisense". "Targeting" an oligonucleotide to a chosen nucleic acid target, in the context of this invention, is a multistep process. The process usually begins with identifying a nucleic acid sequence whose function is to be modulated. This may be, as examples, a cellular gene (or mRNA made from the gene) whose expression is associated with a particular disease state, or a foreign nucleic acid from an infectious agent. In the present invention, the target is a nucleic acid encoding PKC; in other words, a PKC gene or mRNA expressed from a PKC gene. The targeting process also includes determination of a site or sites within the nucleic acid sequence for the oligonucleotide interaction to occur such that the desired effect--modulation of gene expression--will result. Once the target site or sites have been identified, oligonucleotides are chosen which are sufficiently complementary to the target, i.e., hybridize sufficiently well and with sufficient specificity, to give the desired modulation.

In the context of this invention "modulation" means either inhibition or stimulation. Inhibition of PKC gene expression is presently the preferred form of modulation. This modulation can be measured in ways which are routine in the art, for example by Northern blot assay of mRNA expression or Western blot assay of protein expression as taught in the examples of the instant application. Effects on cell proliferation or tumor cell growth can also be measured, as taught in the examples of the instant application.

"Hybridization", in the context of this invention, means hydrogen bonding, also known as Watson-Crick base pairing, between complementary bases, usually on opposite nucleic acid strands or two regions of a nucleic acid strand. Guanine and cytosine are examples of complementary bases which are known to form three hydrogen bonds between them. Adenine and thymine are examples of complementary bases which form two hydrogen bonds between them.

"Specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between the DNA or RNA target and the oligonucleotide.

It is understood that an oligonucleotide need not be 100% complementary to its target nucleic acid sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target interferes with the normal function of the target molecule to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligonucleotide to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment or, in the case of in vitro assays, under conditions in which the assays are conducted.

In the context of this invention, the term "oligonucleotide" refers to a polynucleotide formed from naturally occurring nucleobases and pentofuranosyl (sugar) groups joined by native phosphodiester bonds. This term effectively refers to naturally occurring species or synthetic species formed from naturally occurring subunits or their close homologs.

The term "oligonucleotide" may also refer to moieties which function similarly to naturally occurring oligonucleotides but which have non-naturally occurring portions. Thus, oligonucleotides may have altered sugar moieties, nucleobases or inter-sugar ("backbone") linkages. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, enhanced target binding affinity and increased stability in the presence of nucleases.

Specific examples of some preferred oligonucleotides envisioned for this invention are those which contain intersugar backbone linkages such as phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Most preferred are phosphorothioates and those with CH₂ --NH--O--CH₂, CH₂ --N(CH₃)--O--CH₂ (known as the methylene(methylimino) or MMI backbone), CH₂ --O--N(CH₃)--CH₂, CH₂ --N(CH₃)--N(CH₃)--CH₂ and O--N(CH₃)--CH₂ --CH₂ backbones (where phosphodiester is O--P--O--CH₂). Phosphorothioates are also most preferred. Also preferred are oligonucleotides having morpholino backbone structures. Summerton, J. E. and Weller, D. D., U.S. Pat. No. 5,034,506. In other preferred embodiments, such as the peptide nucleic acid (PNA--referred to by some as "protein nucleic acid") backbone, the phosphodiester backbone of the oligonucleotide may be replaced with a polyamide backbone wherein nucleosidic bases are bound directly or indirectly to aza nitrogen atoms or methylene groups in the polyamide backbone. see, e.g., P. E. Nielsen, M. Egholm, R. H. Berg, O. Buchardt, Science 1991, 254, 1497 and U.S. Pat. No. 5,539,082 and incorporated herein by reference. In accordance with other preferred embodiments, the phosphodiester bonds are substituted with structures which are chiral and enantiomerically specific. Persons of ordinary skill in the art will be able to select other linkages for use in practice of the invention.

Oligonucleotides may also include species which include at least one modified nucleotide base. Thus, purines and pyrimidines other than those normally found in nature may be so employed. Similarly, modifications on the pentofuranosyl portion of the nucleotide subunits may also be effected, as long as the essential tenets of this invention are adhered to. Examples of such modifications are 2'-O-alkyl- and 2'-halogen-substituted nucleotides. Some specific examples of modifications at the 2' position of sugar moieties which are useful in the present invention are OH, SH, SCH₃, F, OCN, O (CH₂)_(n) NH₂ or O(CH₂)_(n) CH₃ where n is from 1 to about 10; C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF₃ ; OCF₃ ; O--, S--, or N-alkyl; O--, S--, or N-alkenyl; SOCH₃ ; SO₂ CH₃ ; ONO₂ ; NO₂ ; N₃ ; NH₂ ; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. One or more pentofuranosyl groups may be replaced by another sugar, by a sugar mimic such as cyclobutyl or by another moiety which takes the place of the sugar.

Chimeric or "gapped" oligonucleotides are also preferred embodiments of the invention. These oligonucleotides contain two or more chemically distinct regions, each comprising at least one nucleotide. Typically, one or more region comprises modified nucleotides that confer one or more beneficial properties, for example, increased nuclease resistance, increased uptake into cells or increased binding affinity for the RNA target. One or more unmodified or differently modified regions retains the ability to direct RNase H cleavage. Chimeric oligonucleotides are disclosed in PCT Publication WO 93/13121 which is assigned to the assignee of the instant application and which is incorporated by reference herein in its entirety. Examples of chimeric oligonucleotides which are presently preferred are 2'-O-methyl or 2'-O-propyl oligonucleotides having a "deoxy gap" region of 2'-deoxynucleotides. Usually this deoxy gap region is located between the two 2'-alkyl regions. In these preferred embodiments, the internucleotide (backbone) linkages may be uniformly phosphorothioate or some combination of phosphorothioate and phosphodiester linkages.

Affinity of an oligonucleotide for its target (in this case a nucleic acid encoding PKC) is routinely determined by measuring the Tm of an oligonucleotide/target pair, which is the temperature at which the oligonucleotide and target dissociate; dissociation is detected spectrophotometrically. The higher the Tm, the greater the affinity of the oligonucleotide for the target. In one embodiment, the region of the oligonucleotide which is modified to increase PKC mRNA binding affinity comprises at least one nucleotide modified at the 2' position of the sugar, most preferably a 2'-O-alkyl or 2'-fluoro-modified nucleotide. Such modifications are routinely incorporated into oligonucleotides and these oligonucleotides have been shown to have a higher Tm (i.e., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target. The effect of such increased affinity is to greatly enhance antisense oligonucleotide inhibition of PKC gene expression. RNAse H is a cellular endonuclease that cleaves the RNA strand of RNA:DNA duplexes; activation of this enzyme therefore results in cleavage of the RNA target, and thus can greatly enhance the efficiency of antisense inhibition. Cleavage of the RNA target can be routinely demonstrated by gel electrophoresis. In another embodiment, the chimeric oligonucleotide is also modified to enhance nuclease resistance. Cells contain a variety of exo- and endo-nucleases which can degrade nucleic acids. A number of nucleotide and nucleoside modifications have been shown to make the oligonucleotide into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide. Nuclease resistance is routinely measured by incubating oligonucleotides with cellular extracts or isolated nuclease solutions and measuring the extent of intact oligonucleotide remaining over time, usually by gel electrophoresis. Oligonucleotides which have been modified to enhance their nuclease resistance survive intact for a longer time than unmodified oligonucleotides. A variety of oligonucleotide modifications have been demonstrated to enhance or confer nuclease resistance. Oligonucleotides which contain at least one phosphorothioate modification are presently more preferred. In some cases, oligonucleotide modifications which enhance target binding affinity are also, independently, able to enhance nuclease resistance.

All such oligonucleotides are best described as being functionally interchangeable with natural oligonucleotides (or synthesized oligonucleotides along natural lines), but having one or more differences from natural structure. All such oligonucleotides are comprehended by this invention so long as they function effectively to hybridize with the PKC RNA.

The oligonucleotides in accordance with this invention preferably comprise from about 5 to about 50 nucleotide units. As will be appreciated, a nucleotide unit is a base-sugar combination (or a combination of analogous structures) suitably bound to an adjacent nucleotide unit through phosphodiester or other bonds forming a backbone structure.

The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for such synthesis may also be employed; the actual synthesis of the oligonucleotides is well within the talents of the routineer. It is also well known to use similar techniques to prepare other oligonucleotides such as phosphorothioates or alkylated derivatives. It is also well known to use similar techniques and commercially available modified amidites and controlled-pore glass (CPG) products such as biotin, fluorescein, acridine or psoralen-modified amidites and/or CPG (available from Glen Research, Sterling, Va.) to synthesize fluorescently labeled, biotinylated or other modified oligonucleotides such as cholesterol-modified oligonucleotides. Other modified and substituted oligomers can be similarly synthesized.

In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information to encode a protein using the three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the 5'-untranslated region, the 3'-untranslated region, the 5' cap region and intron/exon junction ribonucleotides. Thus, oligonucleotides may be formulated in accordance with this invention which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides. In preferred embodiments, the oligonucleotide is specifically hybridizable with a transcription initiation site, a translation initiation site, a 5' cap region, an intron/exon junction, coding sequences or sequences in the 5'- or 3'-untranslated region.

The oligonucleotides of this invention are designed to be hybridizable with messenger RNA derived from the PKC gene. Such hybridization, when accomplished, interferes with the normal roles of the messenger RNA to cause a modulation of its function in the cell. The functions of messenger RNA to be interfered with may include all vital functions such as translocation of the RNA to the site for protein translation, actual translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and possibly even independent catalytic activity which may be engaged in by the RNA. The overall effect of such interference with the RNA function is to modulate expression of the PKC gene.

The oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, and as research reagents and kits. Since the oligonucleotides of this invention hybridize to the PKC gene and its mRNA, sandwich and other assays can easily be constructed to exploit this fact. Furthermore, since the oligonucleotides of this invention hybridize specifically to particular isozymes of the PKC mRNA, such assays can be devised for screening of cells and tissues for particular PKC isozymes. Such assays can be utilized for diagnosis of diseases associated with various PKC forms. Provision of means for detecting hybridization of oligonucleotide with the PKC gene can routinely be accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for detecting the presence or absence of PKC may also be prepared.

The present invention is also suitable for diagnosing abnormal proliferative states in tissue or other samples from patients suspected of having a hyperproliferative disease such as cancer or psoriasis. The ability of the oligonucleotides of the present invention to inhibit cell proliferation may be employed to diagnose such states. A number of assays may be formulated employing the present invention, which assays will commonly comprise contacting a tissue sample with an oligonucleotide of the invention under conditions selected to permit detection and, usually, quantitation of such inhibition. Similarly, the present invention can be used to distinguish PKC-associated tumors, particularly tumors associated with a particular PKC isozyme, from tumors having other etiologies, in order that an efficacious treatment regime can be designed.

The oligonucleotides of this invention may also be used for research purposes. Thus, the specific hybridization exhibited by the oligonucleotides may be used for assays, purifications, cellular product preparations and in other methodologies which may be appreciated by persons of ordinary skill in the art.

The oligonucleotides of the invention are also useful for detection and diagnosis of PKC expression, particularly the specific expression of individual isozymes of PKC. For example, radiolabeled oligonucleotides can be prepared by ³² P labeling at the 5' end with polynucleotide kinase. Sambrook et al., Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Volume 2, p. 10.59. Radiolabeled oligonucleotides are then contacted with tissue or cell samples suspected of PKC expression and the sample is washed to remove unbound oligonucleotide. Radioactivity remaining in the sample indicates bound oligonucleotide (which in turn indicates the presence of PKC) and can be quantitated using a scintillation counter or other routine means. Radiolabeled oligo can also be used to perform autoradiography of tissues to determine the localization, distribution and quantitation of PKC expression for research, diagnostic or therapeutic purposes. In such studies, tissue sections are treated with radiolabeled oligonucleotide and washed as described above, then exposed to photographic emulsion according to routine autoradiography procedures. The emulsion, when developed, yields an image of silver grains over the regions expressing raf. Quantitation of the silver grains permits PKC expression to be detected.

Analogous assays for fluorescent detection of PKC expression can be developed using oligonucleotides of the invention which are conjugated with fluorescein or other fluorescent tag instead of radiolabeling. Such conjugations are routinely accomplished during solid phase synthesis using fluorescently labeled amidites or CPG (e.g., fluorescein-labeled amidites and CPG available from Glen Research, Sterling, Va. See 1993 Catalog of Products for DNA Research, Glen Research, Sterling, Va., p. 21).

Each of these assay formats is known in the art. One of skill could easily adapt these known assays for detection of raf expression in accordance with the teachings of the invention providing a novel and useful means to detect PKC expression, particularly of specific PKC isozymes.

For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with this invention. Oligonucleotides may be formulated in a pharmaceutical composition, which may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the oligonucleotide. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like in addition to oligonucleotides. Conventional chemotherapeutic agents may also be included. Examples of such agents are anticancer and cytotoxic drugs well-known in the art, such as doxorubicin.

The pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be done topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip or by intravenous, subcutaneous, intraperitoneal or intramuscular injection.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Dosing is dependent on severity and responsiveness of the condition to be treated, but will normally be one or more doses per day, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be calculated based on EC50's in in vitro and in vivo animal studies. For example, given the molecular weight of compound (derived from oligonucleotide sequence and chemical structure) and an effective dose such as an IC50, for example (derived experimentally), a dose in mg/kg is routinely calculated. Thus, in the context of this invention, by "therapeutically effective amount" is meant the amount of the compound which is required to have a therapeutic effect on the treated mammal. This amount, which will be apparent to the skilled artisan, will depend upon the type of mammal, the age and weight of the mammal, the type of disease to be treated, perhaps even the gender of the mammal, and other factors which are routinely taken into consideration when treating a mammal with a disease. A therapeutic effect is assessed in the mammal by measuring the effect of the compound on the disease state in the animal. For example, if the disease to be treated is psoriasis, a reduction or ablation of the skin plaque is an indication that the administered dose has a therapeutic effect. Similarly, in mammals being treated for cancer, therapeutic effects are assessed by measuring the rate of growth or the size of the tumor, or by measuring the production of compounds such as cytokines, which production is an indication of the progress or regression of the tumor.

The following examples illustrate the present invention and are not intended to limit the same.

EXAMPLES Example 1 Oligonucleotide Synthesis

Unmodified DNA oligonucleotides were synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry with oxidation by iodine. β-cyanoethyldiisopropyl-phosphoramidites were purchased from Applied Biosystems (Foster City, Calif.). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced by a 0.2M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step was increased to 68 seconds and was followed by the capping step.

2'-O-methyl phosphorothioate oligonucleotides were synthesized according to the procedures set forth above substituting 2'-O-methyl β-cyanoethyldiisopropyl phosphoramidites (Chemgenes, Needham, Mass.) for standard phosphoramidites and increasing the wait cycle after the pulse delivery of tetrazole and base to 360 seconds. Similarly, 2'-O-propyl phosphorothioate oligonucleotides may be prepared by slight modifications of this procedure.

2'-fluoro phosphorothioate oligonucleotides were synthesized using 5'-dimethoxytrityl-3'-phosphoramidites and prepared as described in Example 25 The 2'-fluoro oligonucleotides were prepared using phosphoramidite chemistry and a slight modification of the standard DNA synthesis protocol:deprotection was effected using methanolic ammonia at room temperature.

After cleavage from the controlled pore glass column (Applied Biosystems) and deblocking in concentrated ammonium hydroxide at 55° C. for 18 hours, the oligonucleotides were purified by precipitation twice out of 0.5M NaCl with 2.5 volumes ethanol. Analytical gel electrophoresis was accomplished in 20% acrylamide, 8M urea, 45 mM Tris-borate buffer, Ph 7.0.

The oligonucleotides tested are presented in Table 1. Sequence data are from the CDNA sequence published by Finkenzeller et al., Nucl. Acids Res. 18:2183 (1990); Genbank accession number X52479. The sequence numbers given under the oligonucleotides are relative to the first residue to be sequenced on the CDNA, which is 28 residues upstream of the ATG start codon.

                                      TABLE 1     __________________________________________________________________________     OLIGONUCLEOTIDES TARGETED TO HUMAN PKC-α     SEQ     ID Sequence             Target ISIS #     __________________________________________________________________________     1  CCC           CAA              CCA                 CCT                    CTT                       GCT                          CC 5'     3520        19                1  Untranslated     2  GTT           CTC              GCT                 GGT                    GAG                       TTT                          CA 3'     3521        2063              2044                             Untranslated     3  AAA           ACG              TCA                 GCC                    ATG                       GTC                          CC Translation                                    3522        41                22 init. codon     4  GGA           TTC              ACT                 TCC                    ACT                       GCG                          GG 3'     3526        2109              2090                             Untranslated     5  GAG           ACC              CTG                 AAC                    AGT                       TGA                          TC 3'     3527        2211              2192                             Untranslated     6  CCC           GGG              AAA                 ACG                    TCA                       GCC                          AT Translation                                    3674        47                28 init codon     7  CTG           CCT              CAG                 CGC                    CCC                       TTT                          GC Internal                                    3682        110               91 (C1) domain     8  AGT           CGG              TGC                 AGT                    GGC                       TGG                          AG Internal                                    3686        193               174                             (C1) damain     9  GCA           GAG              GCT                 GGG                    GAC                       ATT                          GA Internal                                    3687        480               461                             (C1) domain     10 GGG           CTG              GGG                 AGG                    TGT                       TTG                          TT 3'     3695        2080              2061                             Untranslated     11 CAC           TGC              GGG                 GAG                    GGC                       TGG                          GG 3'     3875        2098              2079                             Untranslated     12 AGC           CGT              GGC                 CTT                    AAA                       ATT                          TT 3'     3878        2137              2118                             Untranslated     13 ATT           TTC              AGG                 CCT                    CCA                       TAT                          GG 3'     3879        2168              2149                             Untranslated     14 AAG           AGA              GAG                 ACC                    CTG                       AAC                          AG 3'     3884        2217              2198                             Untranslated     15 GAT           AAT              GTT                 CTT                    GGT                       TGT                          AA 3'     3885        2235              2216                             Untranslated     16 ATG           GGG              TGC                 ACA                    AAC                       TGG                          GG Internal                                    3886        2027              2008                             (C3) domain     17 GTC           AGC              CAT                 GGT                    CCC                       CCC                          CC Translation                                    3890        36                17 init. codon     18 CGC           CGT              GGA                 GTC                    GTT                       GCC                          CG Internal                                    3891        63                44 (V1) domain     19 TCA           AAT              GGA                 GGC                    TGC                       CCG                          GC Internal                                    3892        1643              1624                             (C3) domain     20 TGG           AAT              CAG                 ACA                    CAA                       GCC                          GT 3'     3947        2151              2132                             Untranslated     __________________________________________________________________________

Example 2 Cell Culture and Treatment with Phorbol Esters and Oligonucleotides Targeted to PKC-α

PKC protein half-lives have been reported to vary from 6.7 hours to over 24 hours Young et al., Biochem. J. 244:775-779 (1987); Ballester et al., J. Biol. Chem. 260:15194-15199 (1985)!. These long half-lives make inhibiting steady-state levels of PKC-α an unwieldy approach when screening antisense oligonucleotides, due to the long incubation times which would be required. We have therefore made use of the ability of phorbol esters to reversibly lower intracellular levels of PKC. Treatment of cells with phorbol esters causes an initial activation of kinase activity, followed by a down-regulation of PKC. For PKC-α this down-regulation has been shown to be a direct consequence of an increased rate of proteolysis of the kinase with no apparent change in synthetic rate.

We determined that in human lung carcinoma (A549) cells, treatment with the phorbol ester 12,13-dibutyrate (PDBu), using a modification of the method of Krug et al., Krug et al., J. Biol. Chem. 262:11852-11856 (1987)! lowered cellular levels of PKC-α, without affecting PKC-α mRNA levels, and that this effect was reversible. The basis of the assay to screen for potency of oligonucleotides targeting PKC-α is to initially lower PKC-α protein levels by chronic treatment with PDBu, remove PDBu by extensively washing the cells (hence allowing the cells to synthesize fresh PKC-α protein), and incubate the cells with oligonucleotides intended to inhibit the resynthesis of new PKC-α protein.

Procedure: A549 cells (obtained from the American Type Culture Collection, Bethesda Md.) were grown to confluence in 6-well plates (Falcon Labware, Lincoln Park, N.J.) in Dulbecco's modified Eagle's medium (DME) containing 1 g glucose/liter and 10% fetal calf serum (FCS, Irvine Scientific, Santa Ana, Calif.).

Cells were treated with 500 nM PDBu (Sigma Chem. Co., St. Louis, Mo.) for 12-16 hours (overnight). Cells were then washed three times in DME at 37° C., and 1 ml DMA containing 20 μl DOTMA (Lipofectin reagent, BRL, Bethesda, Md.) was added. Oligonucleotides were added to a concentration of 1 μM and the cells were incubated for a further 4 hours at 37° C.

Cells were washed once in 3 ml DME containing 0.1 mg/ml BSA and a further 2 ml DME containing 0.1 mg/ml BSA was added. Oligonucleotides (1 μM) were added and the cells were incubated at 37° C. for 24 hours.

Cells were washed three times in phosphate-buffered saline (PBS) and cellular proteins were extracted in 120 μl sample buffer (60 mM Tris pH 6.8, 2% SDS, 10% glycerol, 10 mM dithiothreitol) and boiled for 5 minutes. Intracellular levels of PKC-α protein were determined by immunoblotting.

Example 3 Immunoblot Assay for PKC Expression

Cell extracts were electrophoresed on 10% SDS-PAGE mini-gels. The resolved proteins were transferred to Immobilon-P membrane (Millipore, Bedford Mass.) by electrophoretic transfer and the membrane was blocked for 60 minutes in TBS (Tris-HCl pH 7.4, 150 mM NaCl) containing 5% nonfat milk. The membrane was then incubated for 16 hours at 4° C. with monoclonal antibodies raised against PKC-α (UBI, Lake Placid, N.Y.) diluted to 0.2 μg/ml in TBS containing 0.2% nonfat milk. This was followed by three washes in TBS plus 0.2% nonfat milk. The membrane was then incubated for one hour with ¹²⁵ I-labelled goat anti-mouse secondary antibody (ICN Radiochemicals, Irvine, Calif.). Membranes were then washed extensively in TBS plus 0.2% nonfat milk. Bands were visualized and quantitated using a Phosphorimager (Molecular Dynamics, Sunnyvale, Calif.). PKC-α appears as a single band with a molecular weight of 80 kD.

Each oligonucleotide was tested three times, in triplicate, and the results of the experiments were normalized against percentage of protein present as compared to cells which were not treated with oligonucleotide (FIGS. 1a and 1b). The five most effective oligonucleotides target the AUG start codon and regions slightly upstream and downstream from it (Sequence Nos. 1, 3, 17, 7, 6). The next most effective oligonucleotides are targeted toward the 3' untranslated region of the RNA (oligos 2, 5, 14).

Example 4 Other Isozymes of PKC

Results with oligonucleotides targeting human PKC-α demonstrated that the most effective target sequences were those surrounding the translation initiation codon and the 3' untranslated region. It is believed that these sequences will also be effective targets for oligonucleotides directed against other isozymes of PKC. The other isozymes of human PKC for which sequence data are available are PKC-β (types I and II), PKC-γ (partial sequence) and PKC-η. Antisense oligonucleotides which are likely to be effective inhibitors of PKC are identified below. These oligonucleotides are synthesized as in Example 1, and can be screened as in Examples 2 and 3, using appropriate antibodies where available. Alternatively, a reporter gene assay system can be established, transiently co-expressing the desired isozyme of PKC with luciferase under the influence of the TPA-responsive enhancer or other suitable promoter. PKC expression is then assayed by measuring luciferase activity using standard procedures. Luciferase is extracted from cells by lysis with the detergent Triton X-100, as described by Greenberg, M. E., in Current Protocols in Molecular Biology, (F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. A. Smith, J. G. Seidman and K. Strahl, eds.), John Wiley and Sons, NY (1987). A Dynatech ML1000 luminometer is used to measure peak luminescence upon addition of luciferin (Sigma) to 625 μM.

PKC-β, types I and II

Sequence data are from Kubo et al., FEBS Lett. 223:138-142 (1987); Genbank accession numbers X06318, M27545, X07109. Sequences are numbered from the first 5' base sequenced on the cDNA. PKC-β types I and II are the result of alternative mRNA splicing of a single gene product. This results in proteins with identical amino termini (5' end of the mRNA); however, there is sequence divergence in the carboxy termini (3' end of the mRNA). The following oligonucleotides, targeted to the translation initiation codon, are expected to modulate expression of both PKC-β types I and II:

                  TABLE 2     ______________________________________     OLIGONUCLEOTIDES TARGETED TO PKC-β TYPES I AND II     SEQ     ID   Sequence                 Target     ______________________________________     21   CAT    CTT    GCG  CGC  GGG  GAG  CC   Translation          139                               120  init.     22   TGC    GCG    CGG  GGA  GCC  GGA  GC   Translation          134                               115  init.     23   CGA    GAG    GTG  CCG  GCC  CCG  GG   Translation          113                               94   init.     24   CTC    TCC    TCG  CCC  TCG  CTC  GG   Translation          183                               164  init.     ______________________________________

The following antisense oligonucleotides are targeted to the 3'-untranslated region of PKC-β type I:

                  TABLE 3     ______________________________________     OLIGONUCLEOTIDES TARGETED TO PKC-β TYPE I     SEQ.     ID   Sequence                 Target     ______________________________________     25   TGG    AGT    TTG  CAT  TCA  CCT  AC   3' Untranslated          2168                              2149     26   AAA    GGC    CTC  TAA  GAC  AAG  CT   "          2285                              2266     27   GCC    AGC    ATG  TGC  ACC  GTG  AA   "          2250                              2231     28   ACA    CCC    CAG  GCT  CAA  CGA  TG   "          2186                              2167     29   CCG    AAG    CTT  ACT  CAC  AAT  TT   "          2569                              2550     ______________________________________

The following antisense oligonucleotides are targeted to the 3'-untranslated region of PKC-β Type II:

                  TABLE 4     ______________________________________     OLIGONUCLEOTIDES TARGETED TO PKC-β TYPE II     SEQ.     ID   Sequence                 Target     ______________________________________     30   ACT    TAG    CTC  TTG  ACT  TCG  GG   3' Untranslated          2160                              2141     31   ATG    CTG    CGG  AAA  ATA  AAT  TG   "          2420                              2401     32   ATT    TTA    TTT  TGA  GCA  TGT  TC   "          2663                              2644     33   TTT    GGG    GAT  GAG  GGT  GAG  CA   "          2843                              2824     34   CCC    ATT    CCC  ACA  GGC  CTG  AG   "          3137                              3118     ______________________________________

PKC-γ

Sequence data are from Coussens et al., Science 233:859-866 (1986); Genbank accession number M13977. Sequences are numbered from the first 5' base sequenced in the cDNA. The full sequence is not available: the extreme 3' end of the open reading frame and the 3' untranslated region are missing. Consequently these regions are not presently available as antisense targets.

                  TABLE 5     ______________________________________     OLIGONUCLEOTIDES TARGETED TO PKC-γ     SEQ.     ID   Sequence                 Target     ______________________________________     35   CGG    AGC    GCG  CCA  GGC  AGG  GA   5' Untranslated          51                                32     36   CCT    TTT    CCC  AGA  CCA  GCC  AT   Translation          215                               196  init.     37   GGC    CCC    AGA  AAC  GTA  GCA  GG   5' of start          195                               176  codon     38   GGA    TCC    TGC  CTT  TCT  TGG  GG   5' Untranslated          170                               151     39   CAG    CCA    TGG  CCC  CAG  AAA  CG   Translation          202                               183  init.     ______________________________________

PKC-η

Sequence data for PKC-η are from Bacher and colleagues Bacher et al., Mol. Cell. Biol. 11:126-133 (1991)!; Genbank accession number M55284. They assign their isozyme the name PKC-L; however the sequence is almost identical to that of mouse PKC-η, so the latter nomenclature is used here for consistency. Sequences are numbered from the first 5' base sequenced in the cDNA.

                  TABLE 6     ______________________________________     OLIGONUCLEOTIDES TARGETED TO PKC-η     SEQ.     ID   Sequence                 Target     ______________________________________     40   CGA    CAT    GCC  GGC  GCC  GCT  GC   Translation          172                               153  init.     41   CAG    ACG    ACA  TGC  CGG  CGC  CG   Translation          176                               157  init.     42   GCC    TGC    TTC  GCA  GCG  GGA  GA   Translation          138                               119  init.     43   ACA    GGT    GCA  GGA  GTC  GAG  GC   Translation          86                                67   init.     44   GTC    CCG    TCT  CAG  GCC  AGC  CC   Translation          111                               92   init.     45   CCT    CAC    CGA  TGC  GGA  CCC  TC   Translation          221                               202  init.     46   ATT    GAA    CTT  CAT  GGT  GCC  AG   Translation          193                               174  init.     47   TCT    CAC    TCC  CCA  TAA  GGC  TA   3' Untranslated          2046                              2027     48   TTC    CTT    TGG  GTT  CTC  GTG  CC   "          2067                              2048     49   TTC    CAT    CCT  TCG  ACA  GAG  TT   "          2353                              2336     50   AGG    CTG    ATG  CTG  GGA  AGG  TC   "          2300                              2281     51   GTT    CTA    AGG  CTG  ATG  CTG  GG   "          2306                              2287     ______________________________________

Example 5 Dose Response of Phosphorothioate/2'-O-methyl Oligonucleotide Effects on PKC-α Protein Synthesis

A series of phosphorothioate, fully 2'-O-methyl oligonucleotides having SEQ ID NO: 1, 2, 3 and 5 were synthesized. A549 cells were treated with 500 nM PDBu for 18 hours to downregulate PKC-α synthesis, washed to remove PDBu and then treated with oligonucleotide and DOTMA/DOPE cationic liposomes. Medium was replaced after four hours and the cells were allowed to recover for another 20 hours. Proteins were extracted and PKC-α protein levels were determined by immunoblotting as described in Example 3. Results were quantified with a phosphorimager (Molecular Dynamics, Sunnyvale, Calif.) and are shown in FIG. 2 expressed as percent of control (saline treatment). ISIS 4649 (SEQ ID NO: 3; squares) reduced PKC-α protein levels by 85-90% at 500 nM and had an IC50 of approximately 260 nM.

Example 6 Effect of Antisense Oligonucleotides on PKC-α mRNA Levels:

A549 cells were treated with phosphorothioate oligonucleotides at 500 nM for four hours in the presence of the cationic lipids DOTMA/DOPE, washed and allowed to recover for an additional 20 hours. Total RNA was extracted and 20 μg of each was resolved on 1.2% gels and transferred to nylon membranes. These blots were probed with a ³² p radiolabeled PKC-α cDNA probe and then stripped and reprobed with a radiolabeled G3PDH probe to confirm equal RNA loading. Each oligonucleotide (3520, 3521, 3522 and 3527) was used in duplicate. The two major PKC-α transcripts (8.5 kb and 4.0 kb) were examined and quantified with a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.). Results are shown in FIG. 3. Oligonucleotides 3521 (SEQ ID NO: 2), 3522 (SEQ ID NO: 3) and 3527 (SEQ ID NO: 5) gave better than 50% reduction of PKC-α mRNA levels. Oligonucleotides 3521 and 3527 gave approximately 80% reduction of the smaller transcript and over 90% reduction of the larger transcript.

Example 7 Chimeric (Deoxy Gapped) 2'-O-methyl Oligonucleotides

Oligonucleotides 3521 (SEQ ID NO: 2), 3522 (SEQ ID NO: 3) and 3527 (SEQ ID NO: 5) were chosen for further study and modification. Oligonucleotides having these sequences were synthesized as uniformly phosphorothioate chimeric oligonucleotides having a centered deoxy gap of various lengths flanked by 2'-O-methylated regions. These oligonucleotides (500 nM concentration) were tested for effects on PKC-α mRNA levels by Northern blot analysis. Results are shown in FIG. 4. Deoxy gaps of eight nucleotides or more gave maximal reduction of PKC-α mRNA levels (both transcripts) in all cases. The oligonucleotide having SEQ ID NO: 3 reduced PKC-α mRNA by approximately 83% with a deoxy gap length of four nucleotides, and gave nearly complete reduction of PKC-α mRNA with a deoxy gap length of six or more.

Dose-response curves for these oligonucleotides are shown in FIG. 5. The 2'-O-methyl chimeric oligonucleotides with four- or six-nucleotide deoxy gaps have an IC50 for PKC-α mRNA reduction (concentration of oligonucleotide needed to give a 50% reduction in PKC-α mRNA levels) of 200-250 nM, as did the full-deoxy oligonucleotide (all are phosphorothioates throughout). The 2'-O-methyl chimeric oligonucleotide with an 8-nucleotide deoxy gap had an IC50 of approximately 85 nM.

Several variations of this chimeric oligonucleotide (SEQ. ID NO: 3) were compared for ability to lower PKC-α mRNA levels. These oligonucleotides are shown in Table 7.

                  TABLE 7     ______________________________________     Chimeric 2'-O-methyl/deoxy P = S oligonucleotides     bold = 2'-O-methyl; s = P = S linkage, o = P = O linkage     OLIGO                            SEQ     #     SEQUENCE                   ID NO:     ______________________________________     3522  AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC                                      3     5352  AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC                                      3     6996  AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC                                      3     7008  AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC                                      3     7024  AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC                                      3     ______________________________________

Effects of these oligonucleotides on PKC-α mRNA levels is shown in FIG. 6. Oligonucleotides 7008, 3522 and 5352 show reduction of PKC-α mRNA, with 5352 being most active.

A series of 2'-O-propyl chimeric oligonucleotides was synthesized having SEQ ID NO: 3. These oligonucleotides are shown in Table 8.

                  TABLE 8     ______________________________________     Chimeric 2'-O-propyl/deoxy P = S oligonucleotides     bold = 2'-O-propyl; s = P = S linkage, o = P = O linkage     OLIGO                            SEQ     #     SEQUENCE                   ID NO:     ______________________________________     7199  AsAsAsAsCsGsTsCsAsGsCsCsAsTsGsGsTsCsCsC                                      3     7273  AoAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCoC                                      3     7294  AsAoAoAoCoGsTsCsAsGsCsCsAsTsGoGoToCoCsC                                      3     7295  AsAoAoAoCoGsToCsAoGsCoCsAsTsGoGoToCoCsC                                      3     ______________________________________

These 2'-O-propyl chimeric oligonucleotides were compared to the 2'-O-methyl chimeric oligonucleotides. Oligonucleotides 7273 and 7294 were more active than their 2'-O-methyl counterparts at lowering PKC-α mRNA levels. This is shown in FIGS. 7 and 8.

Example 8 Additional Oligonucleotides which Decrease PKC-α mRNA

Additional phosphorothioate oligonucleotides targeted to the human PKC-α 3' untranslated region were designed and synthesized. These sequences are shown in Table 9.

                  TABLE 9     ______________________________________     Chimeric 2'-O-propyl/deoxy P = S oligonucleotides     targeted to PKC-α 3'-UTR     bold = 2'-O-propyl; s = P = S linkage, o = P = O linkage     OLIGO #            SEQUENCE                  ID NO:     ______________________________________     6632   TsTsCs TsCsGs CsTsGs GsTsGs AsGsTs TsTsC                                      52     6653   TsTsCs TsCsGs CsTsGs GsTsGs AsGsTs TsTsC                                      52     6665   ToToCo TsCsGs CsTsGs GsTsGs AsGsTo ToToC                                      52     7082   TsCsTs CsGsCs TsGsGs TsGsAs GsTsTs TsC                                      53     7083   TsCsTs CsGsCs TsGsGs TsGsAs GsTsTs TsC                                      53     7084   ToCoTo CsGsCs TsGsGs TsGsAs GsToTo ToC                                      53     ______________________________________

As shown in FIG. 9, oligonucleotides 6632, 6653, 7082 and 7083 are most active in reducing PKC-α mRNA levels.

Example 9 Culture of Human A549 Lung Tumor Cells

The human lung carcinoma cell line A549 was obtained from the American Type Culture Collection (Bethesda, Md.). Cells were grown in Dulbecco's Modified Eagle's Medium (Irvine Scientific, Irvine, Calif.) containing 1 gm glucose/liter and 10% fetal calf serum (Irvine Scientific). Cells were trypsinized and washed and resuspended in the same medium for introduction into mice.

Example 10 Effect of ISIS 3521 on the Growth of Human A549 Lung Tumor Cells in Nude Mice

200 μl of A549 cells (5×10⁶ cells) were implanted subcutaneously in the inner thigh of nude mice. ISIS 3521, a phosphorothioate oligonucleotide with Sequence ID NO 2 was administered twice weekly for four weeks, beginning one week following tumor cell inoculation. Oligonucleotides were formulated with cationic lipids (DMRIE/DOPE) and given subcutaneously in the vicinity of the tumor. Oligonucleotide dosage was 5 mg/kg with 60 mg/kg cationic lipid. Tumor size was recorded weekly.

As shown in FIG. 10, tumor growth was almost completely inhibited in two of the three mice, and reduced compared to control in the third mouse. This inhibition of tumor growth by ISIS 3521 is statistically significant. The control oligonucleotide (ISIS 1082) is a 21-mer phosphorothioate oligonucleotide without significant sequence homology to the PKC mRNA target.

Administration of oligonucleotides to mice whose tumors had already reached detectable size had no discernable effect on subsequent tumor growth.

Example 11 Effect of Antisense Oligonucleotides on Growth of Human MDA-MB231 Tumors in Nude Mice

MDA-MB231 human breast carcinoma cells were obtained from the American Type Culture Collection (Bethesda, Md.). Serially transplanted MDA-MB231 tumors were established subcutaneously in nude mice. Beginning two weeks later, oligonucleotides 3521 and 3527, a phosphorothioate oligonucleotide having Sequence ID NO. 5, in saline, were administered intravenously daily for 14 days at dosages of 60 mg/kg and 6 mg/kg. Control oligonucleotide ISIS 1082 was also administered at these doses, and a saline control was also given. Tumor growth rates wre monitored for the two-week period of oligonucleotide administration. As shown in FIG. 11, both PKC-α oligonucleotides (3521 and 3527) significantly inhibit tumor growth at dosages of 60 mg/kg and 6 mg/kg. The control oligonucleotide (ISIS 1082) also showed some reduction in tumor growth, but this effect was less than with antisense oligonucleotides even at high doses, and considerably less at the lower dose. A lower-dose study was conducted using the same oligonucleotides at 6 mg/kg and 0.6 mg/kg. At 0.6 mg/kg ISIS 3521 significantly reduced tumor growth. At this concentration, ISIS 3527 also reduced tumor growth, but this result was not statistically significant.

Example 12 Effect of Oligonucleotides on the Growth of Murine Lewis Lung Carcinoma in Mice

Serially transplanted murine Lewis lung carcinomas were established in mice. Oligonucleotides 3521 and 3527 were administered intravenously every day for 14 days at doses of 6 mg/kg and 0.6 mg/kg. Tumor growth rates were monitored for the two-week period of oligonucleotide administration. As expected, these oligonucleotides, which are targeted to human PKC sequences, had insignificant effects on the mouse-derived tumors.

Example 13 Effects of Antisense Oligonucleotide ISIS 4189 on endogenous PKC-α expression in hairless mice

In order to study oligonucleotide effects on endogenous PKC mRNA levels in normal animals, it was necessary to employ an oligonucleotide complementary to the murine PKC-α. ISIS 4189 is a 20-mer phosphorothioate oligonucleotide targeted to the AUG codon of mouse PKC-α. This region is without homology to the human PKC sequence and the oligonucleotide has no effect on expression of PKC-α in human cells. ISIS 4189 has an IC50 of 200 nM for mRNA reduction in C127 mouse breast epithelial cells. ISIS 4189 in saline was administered intraperitoneally to hairless mice at concentrations of 1, 10 or 100 mg/kg body weight. Injections were given daily for seven days. Tissues from liver, kidney, spleen, lung and skin were removed and PKC-α mRNA and protein levels were determined. Histopathological examination was also performed on liver, kidney and lung samples. ISIS 4189 at 100 mg/kg inhibited endogenous PKC-α mRNA levels in the mouse liver to 10-15% of control (saline) levels.

Example 14 Screening of Antisense Oligonucleotides Targeted to Human PKC-η

A series of 20-mer phosphorothioate oligonucleotides complementary to human PKC-η were synthesized. These oligonucleotides were screened at a concentration of 500 nM for ability to decrease PKC-η mRNA levels in human A549 cells, using a Northern blot assay. The oligonucleotide sequences are shown in Table 10 and the results are shown in FIG. 12.

                                      TABLE 10     __________________________________________________________________________     OLIGONUCLEOTIDES TARGETED TO HUMAN PKC-η mRNA     SEQ ID         Sequence              Target                                    ISIS #     __________________________________________________________________________     6431         CGA            CAT               GCC                  GGC                     GCC                        GCT                           GC  AUG  40     6442         CAG            ACG               ACA                  TGC                     CGG                        CGC                           CG  AUG  41     6443         GCC            TGC               TTC                  GCA                     GCG                        GGA                           GA  5' UTR                                    42     6432         ACA            GGT               GCA                  GGA                     GTC                        GAG                           GC  5' UTR                                    43     6433         GTC            CCG               TCT                  CAG                     GCC                        AGC                           CC5'                               UTR  44     6435         CCT            CAC               CGA                  TGC                     GGA                        CCC                           TC  Coding                                    45     6441         ATT            GAA               CTT                  CAT                     GGT                        GCC                           AG  Coding                                    46     6581         TCT            CAC               TCC                  CCA                     TAA                        GGC                           TA  3' UTR                                    47     6580         TTC            CTT               TGG                  GTT                     CTC                        GTG                           CC  3' UTR                                    48     6436         AAC            TCG               AGG                  TGG                     CCG                        CCG                           TC  Coding                                    54     6434         CGC            CTT               CGC                  ATA                     GCC                        CTT                           TG  Coding                                    55     6444         GGA            AGG               GGT                  GAT                     TGC                        GGG                           CC  Coding                                    56     6445         AAC            ACG               CCC                  ATT                     GCC                        CAC                           CA  Coding                                    57     6446         GTC            TCA               AGA                  TGG                     CGT                        GCT                           CG  Coding                                    58     6553         GCG            ATG               GTT                  CAG                     CTG                        GGC                           CC  Coding                                    59     6605         GCC            CTC               TCT                  CTC                     ACT                        CCC                           CA  3' UTR                                    60     6579         CTG            GGA               AGG                  TCC                     GAT                        AGA                           GG  3' UTR                                    61     6603         AAG            GCT               GAT                  GCT                     GGG                        AAG                           GT  3' UTR                                    62     __________________________________________________________________________

Oligonucleotides 6432, 6443, 6431, 6442, 6435, 6434, 6445, 6553, 6581 and 6603 reduced PKC-η mRNA levels by greater than 50%. The most potent oligonucleotides were ISIS 6581 (targeting 3' untranslated region) and ISIS 6445 (targeting coding region) which gave nearly complete loss of PKC mRNA in this assay.

Example 15 Screening of Antisense Oligonucleotides Targeted to PKC-δ

A series of 20-mer phosphorothioate oligonucleotides complementary to human PKC-δ were synthesized. These oligonucleotides were screened by Northern blot assay at a concentration of 500 nM for their ability to decrease PKC-δ mRNA levels in human A549 cells. The oligonucleotide sequences and the results, expressed as percent inhibition of PKC-δ mRNA expression, are shown in Table 11.

                                      TABLE 11     __________________________________________________________________________     Antisense oligonucleotides targeted to PKC-δ     Isis #         Site             Sequence             % Inhib                                      SEQ ID:     __________________________________________________________________________     10299         AUG GCA                GGA                   ACG                      GCG                         CCA                            TGG                               TG  0% 63     10300         Coding             CTG                GTT                   CGC                      CTC                         GTC                            CTC                               GG 25  64     10301         Coding             ATC                TGG                   ATG                      ACG                         CGC                            CCC                               TC 26  65     10302         Coding             TTC                TTG                   CAG                      CGC                         TCG                            GCC                               AG  8  66     10303         Coding             TGC                AAT                   CCA                      CGT                         CCT                            CCA                               GG 50  67     10304         Coding             GGC                TCC                   GCG                      GCG                         GTT                            CAT                               CG 12  68     10305         Coding             AAG                CGG                   TGC                      GGC                         ATG                            TCG                               AT 43  69     10306         Coding             GCA                GGC                   TGC                      CGC                         AGT                            GGT                               CA 12  70     10307         Coding             CCT                CCC                   CAG                      CAA                         CTC                            CGG                               TC 36  71     10308         Coding             AGC                GGC                   CTT                      TGT                         CCT                            GGA                               TG 11  72     10309         Coding             GGC                CAT                   CCC                      GGT                         CCA                            ACA                               GC 43  73     10310         Coding             GGT                GCT                   GGC                      CCG                         GCT                            CTC                               CC 66  74     10311         Coding             GGA                CCC                   CGA                      AAG                         ACC                            ACC                               AG 77  75     10312         Coding             GTG                GCT                   CCA                      ACC                         TCC                            GCT                               TT 18  76     10313         Coding             AGG                AGG                   TGC                      TCG                         AAT                            TTG                               GG  0% 77     __________________________________________________________________________

Oligonucleotides ISIS 10303, ISIS 10310 and ISIS 10311 gave at least 50% inhibition of PKC-δ mRNA expression in this assay and are preferred.

Example 16 Screening of Aantisense Oligonucleotides Targeted to PKC-ε

A series of 20-mer phosphorothioate oligonucleotides complementary to human PKC-ε were synthesized. These oligonucleotides were screened by Northern blot assay at a concentration of 500 nM for their ability to decrease PKC-ε mRNA levels in human A549 cells. The oligonucleotide sequences and the results, expressed as percent inhibition of PKC-ε mRNA expression, are shown in Table 12.

                                      TABLE 12     __________________________________________________________________________     Antisense oligonucleotides targeted to PKC-ε     Isis #        Site            Sequence             % Inhib                                     SEQ ID:     __________________________________________________________________________     7933        AUG ACT               ACC                  ATG                     GTC                        GGG                           GCG                              GG  0% 78     7934        Coding            GTC               CCA                  CCG                     CAT                        GGC                           GCA                              GC  0  79     7935        Coding            GTT               TGG                  CCG                     ATG                        CGC                           GAG                              TC  0  80     7936        Coding            TGC               AGT                  TGG                     CCA                        CGA                           AGT                              CG  0  81     8032        Coding            GTG               GGG                  CAT                     GTT                        GAC                           GCT                              GA  0  82     8031        Coding            CCA               GAG                  CAG                     GGA                        CCC                           ACA                              GT  0  83     7939        Coding            TCT               CCT                  CGG                     TTG                        TCA                           AAT                              GA  0  84     7940        Coding            CGG               TGC                  TCC                     TCT                        CCT                           CGG                              TT  0  85     7941        Coding            AGC               CAA                  AAT                     CCT                        CTT                           CTC                              TG  0  86     7942        Coding            CAT               GAG                  GGC                     CGA                        TGT                           GAC                              CT 67  87     7943        Coding            ATC               CCT                  TCC                     TTG                        CAC                           ATC                              CC  3  88     7944        Coding            CCC               CAG                  GGC                     CCA                        CCA                           GTC                              CA 38  89     7945        Coding            AGC               ACC                  CCC                     AGG                        GCC                           CAC                              CA 42  90     7946        Coding            CGT               ACA                  TCA                     GCA                        CCC                           CCA                              GG 42  91     7947        Coding            CCA               GCC                  ATC                     ATC                        TCG                           TAC                              AT  9  92     7948        Coding            TGC               CAC                  ACA                     GCC                        CAG                           GCG                              CA 55  93     7949        Stop            TCA               GGG                  CAT                     CAG                        GTC                           TTC                              AC  0  94     7950        Stop            CTC               TCA                  GGG                     CAT                        CAG                           GTC                              TT  0  95     __________________________________________________________________________

Oligonucleotides ISIS 7942 and ISIS 7948 gave at least 50% inhibition of PKC-ε mRNA expression in this assay and are preferred.

Additional oligonucleotides targeted to PKC-ε were synthesized. These are shown in Table 13.

                  TABLE 13     ______________________________________     Antisense oligonucleotides targeted to PKC-ε                                           SEQ     Isis #          Site    Sequence                 ID:     ______________________________________     A    Coding  AAG    GAA  AGT  CTG  CGG  CCG  GG    96     B    Coding  TGG    CGG  CTC  CCG  TTC  TGC  AG    97     C    Coding  GCT    TCC  TCG  GCC  GCA  TGC  GT    98     D    Coding  TTG    ACG  CTG  AAC  CGC  TGG  GA    99     E    Coding  GCC    CGG  TGC  TCC  TCT  CCT  CG   100     F    Coding  GGG    CCG  ATG  TGA  CCT  CTG  CA   101     G    Coding  TGG    AGG  AAC  ATG  AGG  GCC  GA   102     H    Coding  CCC    CCA  GGG  CCC  ACC  AGT  CC   103     I    Coding  TGC    GAT  GCC  ACA  CAG  CCC  AG   104     J    Stop    TGG    GCT  CTC  AGG  GCA  TCA  GG   105     ______________________________________

Example 17 Screening of Antisense Oligonucleotides Targeted to PKC-ζ

A series of 20-mer phosphorothioate oligonucleotides complementary to human PKC-ζ were synthesized. These oligonucleotides were screened by Northern blot assay at a concentration of 500 nM for their ability to decrease PKC-ζ mRNA levels in human A549 cells. The oligonucleotide sequences and the results, expressed as percent inhibition of PKC-ζ mRNA expression, are shown in Table 14.

                                      TABLE 14     __________________________________________________________________________     Antisense oligonucleotides targeted to PKC-ζ     Isis #        Site            Sequence             % Inhib                                     SEQ ID:     __________________________________________________________________________     9007        AUG CGC               CGC                  TCC                     CTT                        CCA                           TCT                              TG 67% 106     9008        Coding            CCC               CGT                  AAT                     GCG                        CCT                           TGA                              GG 64  107     9009        Coding            CTG               TCC                  ACC                     CAC                        TTG                           AGG                              GT 14  108     9012        Coding            TTG               GAA                  GAG                     GTG                        GCC                           GTT                              GG 78  109     9013        Coding            CCT               GTT                  AAA                     GCG                        CTT                           GGC                              TT 67  110     9014        Coding            TGC               AGG                  TCA                     GCG                        GGA                           CGA                              GG 40  111     9016        Coding            AGC               CCC                  TGA                     GAG                        ATT                           TTG                              AT  0  112     9017        Coding            TTC               TTC                  AAC                     CGC                        ACC                           AGG                              AG 67  113     9019        Coding            TCC               TTG                  CAC                     ATG                        CCG                           TAG                              TC 24  114     9021        Coding            GGA               GCG                  CCC                     GGC                        CAT                           CAT                              CT 78  115     9022        Coding            GGG               CTC                  GCT                     GGT                        GAA                           CTG                              TG 85  116     9023        3' UTR            GAC               GCA                  CGC                     GGC                        CTC                           ACA                              CC 88  117     9025        3' UTR            TCG               GAG                  CCG                     TGC                        CCA                           GCC                              TG 88  118     9026        3' UTR            CGG               GCC                  AGG                     TGT                        GAG                           GGA                              CT 40  119     9027        3' UTR            CCG               CGA                  CGC                     AGG                        CAC                           AGC                              AG 40  120     9029        3' UTR            GGT               CAG                  TGC                     ATC                        GAG                           TTC                              TG 77  121     __________________________________________________________________________

Oligonucleotides ISIS 9007, 9008, 9012, 9013, 9017, 9021, 9022, 9023, 9025 and 9029 gave greater than 50% inhibition of PKC-ζ mRNA expression in this assay and are preferred. Of these, ISIS 9022, ISIS 9023 and ISIS 9025 gave at least 85% inhibition and are more preferred.

Example 18 Effect of ISIS 3521 on the Growth of Human T24 Bladder Tumors in Nude Mice

Subcutaneous human T24 bladder carcinoma xenografts in nude mice were established by injection of 5×10⁶ T24 cells under the skin. Mice were treated with ISIS 3521 or ISIS 4559, a phosphorothioate scrambled version of the ISIS 3521 sequence, or ISIS 1082, an unrelated control phosphorothioate oligonucleotide targeted to Herpes simplex virus (oligonucleotide doses 0.006 mg/kg, 0.06 mg/kg, 0.6 mg/kg or 6.0 mg/kg) or saline administered intraperitoneally three times weekly. By day 21, ISIS 1082 or ISIS 4559 had no effect on tumor growth at any dose. By day 21, ISIS 3521 showed a dose-dependent inhibition of tumor growth at all dose levels, with a maximal inhibition of 90% at the 6 mg/kg dose.

Example 19 Effect of ISIS 3521 on the Growth of Human Colo-205 Colon Tumors in Nude Mice

Subcutaneous human Colo-205 colon carcinoma xenografts in nude mice were established by injection of 5×10⁶ Colo-205 cells under the skin. Mice were treated with ISIS 3521 and an unrelated control phosphorothioate oligonucleotide (ISIS 1082) administered intravenously once per day at a dosage of 6.0 mg/kg. In this study, ISIS 3521 inhibited tumor growth after 25 days by 84% compared to saline controls. The control oligonucleotide, ISIS 1082, inhibited tumor growth by 20%.

Example 20 Effect of ISIS 8469, a 2'-fluoro Gapped Version of ISIS 3521, on the Growth of A549 Human Lung Tumors in Nude Mice

Subcutaneous human A549 lung adenocarcinoma xenografts were established by injection of 5×10⁶ A549 cells under the skin of Balb/c nude mice. Mice were treated with ISIS 8469, a chimeric version of ISIS 3521 having an 8-nucleotide deoxy gap flanked by six 2'-fluoro nucleotides on each side, as in Example 10. Oligonucleotide doses were from 0.006 mg/kg to 6.0 mg/kg. ISIS 8469 decreased tumor size at all doses in a dose-dependent manner, as shown in FIG. 13. This compound is therefore preferred.

Example 21 U-87 Human Glioblastoma Cell Culture and Subcutaneous Xenografts into Nude Mice

The U-87 human glioblastoma cell line was obtained from the ATCC (Rockville, Md.) and maintained in Iscove's DMEM medium supplemented with heat-inactivated 10% fetal calf serum. Nude mice were injected subcutaneously with 2×10⁷ cells. Mice were injected intraperitoneally with ISIS 3521 at dosages of either 2 mg/kg or 20 mg/kg for 21 consecutive days beginning 7 days after xenografts were implanted. Tumor volumes were measured on days 14, 21, 24, 31 and 35. On day 35 (7 days after end of treatment), ISIS 3521 at 2 mg/kg had reduced tumor volume by 84% compared to saline or sense oligonucleotide control. The 20 mg/kg dose reduced tumor size by 91% on day 35.

Example 22 Effect of ISIS 3521 on PKC-α Protein Levels in U-87 Glioblastoma Xenografts in Nude Mice

PKCα protein levels in subcutaneous U-87 tumor xenografts were measured by western blot analysis on day 24 (day 17 of treatment with ISIS 3521) and day 35 (7 days after end of treatment with ISIS 3521). An affinity-purified PKCα-specific polyclonal antibody (Life Technologies, Inc.) was used as the primary antibody. By day 24, ISIS 3521 was found to virtually totally abolish PKCα in the tumors. By seven days after cessation of oligonucleotide treatment (day 35), PKCα had returned to control levels.

Example 23 "Crossover Experiment" to Evaluate Effect of Switching Treatment on Tumor Size

The two groups of mice with subcutaneous U-87 xenografts previously treated with ISIS 3521 (2 mg/kg or 20 mg/kg) were switched to different treatments on day 35 (7 days after the initial 21 day treatment had ended). The group which had previously received 20 mg/kg ISIS 3521 now received saline ("high dose-to-control"). The group which had received 2 mg/kg ISIS 3521 now received 20 mg/kg ISIS 3521 ("low dose-to-high dose"). This crossover treatment was continued for 21 days as for the original treatment. As shown in FIG. 14, the growth of the tumors in the "low dose-to-high dose" group (open triangles) leveled off after treatment was switched (arrow). The growth of the tumors in the "high dose-to-control" group (closed triangles) rapidly accelerated after switching to saline treatment (arrow). S=sense oligonucleotide (control); AS=antisense oligonucleotide (ISIS 3521) targeted to PKCα.

Example 24 Effect of ISIS 3521 on Intracerebral U-87 Glioblastoma Xenografts into Nude Mice

U-87 cells how were implanted in the brains of nude mice. Mice were treated via continuous intraperitoneal administration of antisense oligonucleotide ISIS 3521 (20 mg/kg), control sense oligonucleotide (20 mg/kg) or saline beginning on day 7 after xenograft implantation. All mice survived until day 25, at which point the saline-treated mice began to die. All saline-treated mice and sense oligonucleotide-treated mice were dead by day 41. In contrast, all ISIS 3521-treated mice were alive until approximately day 37, and half of the mice were still alive at day 61. At the termination of the experiment at day 80, 400%. of the ISIS 3521-treated mice were still alive.

Example 25 Preparation of 2'-Deoxy-2'-fluoro Modified Oligonucleotides

A. N⁶ -Benzoyl- 2'-deoxy-2'-fluoro-5'-O-(4,4'-di-methoxy trityl)! adenosine-3'-O-(N,N-diisopropyl -β-cyanoethyl phosphoramidite.

N⁶ -Benzoyl-9-(2'-fluoro-b-D-ribofuranosyl)adenine was prepared from 9-b-D-arabinofuranosyladenine in a five-step synthesis using a modification of a procedure reported by M. Ikehara at al., Nucleosides and Nucleotides 2:373-385 (1983). Thus, the N⁶ -benzoyl derivative was obtained in good yield utilizing the method of transient protection with chlorotrimethylsilane. R. A. Jones, J. Am. Chem. Soc. 104:1316 (1982). Selective protection of the 3' and 5'-hydroxyl groups of N⁶ -Benzoyl-9-β-D-arabinofuranosyladenine with tetrahydro-pyranyl (THP) was accomplished by modification of a literature procedure G. Butke, et al., in Nucleic Acid Chemistry, Part 3:149-152, Townsend, L. B. and Tipson, R. S. eds., (J. Wiley and Sons, New York 1986) to yield N⁶ -Benzoyl-9- 3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabino furanosyl!adenine in good yield. Treatment of N⁶ -Benzoyl-9- 3',5'-di-O-tetrahydro-pyran-2-yl)-β-D-arabinofuranosyl!adenine with trifluoromethanesulfonic anhydride in dichloromethane gave the 2'-tri-flate derivative N⁶ -Benzoyl-9- 2'-O-trifluoromethylsulfonyl-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabino furanosyl!adenine which was not isolated due to its lability. Displacement of the 2'-triflate group was effected by reaction with tetra-butylammonium fluoride in tetrahydrofuran to obtain a moderate yield of the 2'-fluoro derivative N⁶ -Benzoyl-9- 2'-fluoro-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabino furanosyl!-adenine. Deprotection of the THP groups of N⁶ -Benzoyl-9- 2'-fluoro-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabino-furanosyl!adenine was accomplished by treatment with Dowex-50W in methanol to yield N⁶ -benzoyl-9-(2'-deoxy-2'-fluoro-β-D-ribofuranosyl)adenine in moderate yield. The ¹ H-NMR spectrum of 6 was in agreement with the literature values. M. Ikehara and H. Miki, Chem. Pharm. Bull. 26:2449-2453 (1978). Standard methodologies were employed to obtain the 5'-dimeth-oxytrityl-3'-phosphoramidite intermediates N⁶ -Benzoyl-9- 2'-fluoro-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl!adenine and N⁶ -Benzoyl- 2'-deoxy-2'-fluoro -5'-O-(4,4'-dimethoxytrityl)!adenosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite. K. K. Ogilvie, Can J. Chem. 67:831-839 (1989).

B. N⁶ -Benzoyl-9-β-D-arabinofuranosyladenine.

9-β-D-arabinofuranosyladenine (1.07 g, 4.00 m.mol) was dissolved in anhydrous pyridine (20 mL) and anhydrous dimethylformamide (20 ml) under an argon atmosphere. The solution was cooled to ice temperature and chlorotrimethyl-silane (3.88 ml, 30.6 m.mol) was added slowly to the reaction mixture via syringe. After stirring the reaction mixture at ice temperature for 30 minutes, benzoyl chloride (2.32 ml, 20 m.mol) was added slowly. The reaction mixture was allowed to warm to 20° C. and stirred for 2 hours. After cooling the reaction mixture to ice temperature, cold water (8 ml) was added and the mixture was stirred for 15 minutes. Concentrated ammonium hydroxide (8 ml) was slowly added to the reaction mixture to give a final concentration of 2M of ammonia. After stirring the cold reaction mixture for 30 minutes, the solvent was evaporated in vacuo (60 torr) at 20° C. followed by evaporation in vacuo (1 torr) at 40° C. to give an oil. This oil was triturated with diethyl ether (50 ml) to give a solid which was filtered and washed with diethyl ether three times. This crude solid was triturated in methanol (100 ml) at reflux temperature three times and the solvent was evaporated to yield N⁶ -Benzoyl-9-β-D-arabino-furanosyladenine as a solid (1.50 g, 100%).

C. N⁶ -Benzoyl-9- 3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabino furansoyl!adenine.

N⁶ -Benzoyl-9-β-D-arabinofuranosyladenine (2.62 g, 7.06 m.mol) was dissolved in anhydrous dimethylformamide (150 ml) under an argon atmosphere and p-toluenesulfonic acid monohydrate (1.32 g, 6.92 m.mol) was added. This solution was cooled to ice temperature and dihydropyran (1.26 ml, 13.8 m.mol) was added via syringe. The reaction mixture was allowed to warm to 20° C. Over a period of 5 hours a total of 10 equivalents of dihydropyran were added in 2 equivalent amounts in the fashion described. The reaction mixture was cooled to ice temperature and saturated aqueous sodium bicarbonate was added slowly to a pH of 8, then water was added to a volume of 750 ml. The aqueous mixture was extracted with methylene chloride four times (4×200 ml), and the organic phases were combined and dried over magnesium sulfate. The solids were filtered and the solvent was evaporated in vacuo (60 torr) at 30° C. to give a small volume of liquid which was evaporated in vacuo (1 torr) at 40° C. to give an oil. This oil was coevaporated with p-xylene in vacuo at 40° to give an oil which was dissolved in methylene chloride (100 ml). Hexane (200 ml) was added to the solution and the lower-boiling solvent was evaporated in vacuo at 30° C. to leave a white solid suspended in hexane. This solid was filtered and washed with hexane three times (3×10 ml) then purified by column chromatography using silica and methylene chloride-methanol (93:7, v/v) as eluent. The first fraction yielded the title compound 3 as a white foam (3.19 g, 83%) and a second fraction gave a white foam (0.81 g) which was characterized as the 5'-mono-tetrahydropyranyl derivative of N⁶ -Benzoyl-9-β-D-arabinofuranosyladenine.

D. N⁶ -Benzoyl-9- 2'-O-trifluoromethylsulfonyl-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine.

N⁶ -Benzoyl-9- 3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine (2.65 g, 4.91 m.mol) was dissolved in anhydrous pyridine (20 ml) and the solvent was evaporated in vacuo (1 mm Hg) at 40° C. The resulting oil was dissolved in anhydrous methylene chloride (130 ml) under an argon atmosphere and anhydrous pyridine (3.34 ml, 41.3 m.mol) and N,N-dimethylaminopyridine (1.95 g, 16.0 mmol) were added. The reaction mixture was cooled to ice temperature and trifluoromethanesulfonic anhydride (1.36 ml, 8.05 mmol) was added slowly via syringe. After stirring the reaction mixture at ice temperature for 1 h, it was poured into cold saturated aqueous sodium bicarbonate (140 ml). The mixture was shaken and the organic phase was separated and kept at ice temperature. The aqueous phase was extracted with methylene chloride two more times (2×140 ml). The organic extracts which were diligently kept cold were combined and dried over magnesium sulfate. The solvent was evaporated in vacuo (60 torr) at 20° C. then evaporated in vacuo (1 torr) at 20° C. to give N⁶ -Benzoyl-9- 2'-O-trifluoromethylsulfonyl-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine as a crude oil which was not purified further.

E. N⁶ -Benzoyl-9- 2'-fluoro-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine.

N⁶ -Benzoyl-9- 2'-O-trifluoromethylsulfonyl-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine (<4.9 mmol) as a crude oil was dissolved in anhydrous tetrahydro-furan (120 ml) and this solution was cooled to ice temperature under an argon atmosphere. Tetrabutylammonium fluoride as the hydrate (12.8 g, 49.1 mmol) was dissolved in anhydrous tetrahydrofuran (50 ml) and half of this volume was slowly added via syringe to the cold reaction mixture. After stirring at ice temperature for 1 hour, the remainder of the reagent was added slowly. The reaction mixture was stirred at ice temperature for an additional 1 hour, then the solvent was evaporated in vacuo (60 torr) at 20° C. to give an oil. This oil was dissolved in methylene chloride (250 ml) and washed with brine three times. The organic phase was separated and dried over magnesium sulfate. The solids were filtered and the solvent was evaporated to give an oil. The crude product was purified by column chromatography using silica in a sintered-glass funnel (600 ml) and ethyl acetate was used as eluent. N⁶ -Benzoyl-9- 2'-fluoro-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine was obtained as an oil (2.03 g, 76%).

F.N⁶ -Benzoyl-9-(2'-fluoro-β-D-ribofuranosyl)adenine.

N⁶ -Benzoyl-9- 2'-fluoro-3',5'-di-O-tetrahydropyran-2-yl)-β-D-arabinofuranosyl!adenine (1.31 g, 2.42 mmol) was dissolved in methanol (60 ml), and Dowex 50 W×2-100 (4 cm3, 2.4 m.eq) was added to the reaction mixture. The reaction mixture was stirred at 20° C. for 1 hour then cooled to ice temperature. Triethylamine (5 ml) was then slowly added to the cold reaction mixture to a pH of 12. The resin was filtered and washed with 30% triethylamine in methanol until the wash no longer contained UV absorbing material. Toluene (50 ml) was added to the washes and the solvent was evaporated at 24° C. in vacuo (60 torr then 1 torr) to give a residue. This residue was partially dissolved in methylene chloride (30 ml) and the solvent was transferred to a separatory funnel. The remainder of the residue was dissolved in hot (60° C.) water and after cooling the solvent it was also added to the separatory funnel. The biphasic system was extracted, and the organic phase was separated and extracted three times with water (3×100 ml). The combined aqueous extracts were evaporated in vacuo (60 torr then 1 torr Hg) at 40° C. to give an oil which was evaporated with anhydrous pyridine (50 ml). This oil was further dried in vacuo (1 torr Hg) at 20° C. in the presence of phosphorous pentoxide overnight to give N⁶ -Benzoyl 9-(2'-fluoro-b-D-ribofuranosyl)adenine as a yellow foam (1.08 g, 100%) which contained minor impurities.

G. N⁶ -Benzoyl-9- 2'-fluoro-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl!adenine.

N⁶ -Benzoyl-9-(2'-fluoro-b-D-ribofuranosyl)adenine (1.08 g, 2.89 mmol) which contained minor impurities was dissolved in anhydrous pyridine (20 ml) under an argon atmosphere, and dry triethylamine (0.52 ml, 3.76 mmol) was added followed by addition of 4,4'-dimethoxytrityl chloride (1.13 g, 3.32 mmol). After 4 hours of stirring at 20° C. the reaction mixture was transferred to a separatory funnel and diethyl ether (40 ml) was added to give a white suspension. This mixture was washed with water three times (3×10 ml), the organic phase was separated and dried over magnesium sulfate. Triethylamine (1 ml) was added to the solution and the solvent was evaporated in vacuo (60 torr Hg) at 20° C. to give an oil which was evaporated with toluene (20 ml) containing triethylamine (1 ml). This crude product was purified by column chromatography using silica and ethyl-acetate-triethylamine (99:1, v/v) followed by ethyl acetate-methanol-triethylamine (80:19:1) to give the product in two fractions. The fractions were evaporated in vacuo (60 torr then 1 torr Hg) at 20° C. to give a foam which was further dried in vacuo (1 torr Hg) at 20° C. in the presence of sodium hydroxide to give N⁶ -Benzoyl-9- 2'-fluoro-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl!adenine as a foam (1.02 g, 52%).

H. N⁶ -Benzoyl- 2'-fluoro-5'-O-(4,4'-dimethoxy trityl)!adenosine-3'-O-N,N-diisopropyl-β-cyanoethyl phosphoramidite.

N⁶ -Benzoyl-9- 2'-fluoro-5'-O-(4,4'-dimethoxytrityl)-β-D-ribofuranosyl!adenine (1.26 g, 1.89 mmol) was dissolved in anhydrous dichloromethane (13 ml) under an argon atmosphere, diisopropylethylamine (0.82 ml, 4.66 mmol) was added, and the reaction mixture was cooled to ice temperature. Chloro(diisopropylamino)-β-cyanoethoxyphosphine (0.88 ml, 4.03 mmol) was added to the reaction mixture which was allowed to warm to 20° C. and stirred for 3 hours. Ethylacetate (80 ml) and triethylamine (1 ml) were added and this solution was washed with brine solution three times (3×25 ml). The organic phase was separated and dried over magnesium sulfate. After filtration of the solids the solvent was evaporated in vacuo at 20° C. to give an oil which was purified by column chromatography using silica and hexane-ethyl acetate-triethyl-amine (50:49:1) as eluent. Evaporation of the fractions in vacuo at 20° C. gave a foam which was evaporated with anhydrous pyridine (20 ml) in vacuo (1 torr) at 26° C. and further dried in vacuo (1 torr Hg) at 20° C. in the presence of sodium hydroxide for 24 h to give N⁶ -Benzoyl- 2'-deoxy-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)!adenosine-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite as a foam (1.05 g, 63%).

I. 2'-Deoxy-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)uridine-3'-O-(N,N-diisopropyl-β-cyanoethyl phosphoramidite).

2,2'-Cyclouridine is treated with a solution of 70% hydrogen fluoride/pyridine in dioxane at 120° C. for ten hours to provide after solvent removal a 75% yield of 2'-deoxy-2'-fluorouridine. The 5'-DMT and 3'-cyanoethoxydiisopropylphos-phoramidite derivitized nucleoside is obtained by standard literature procedures, M. J. Gait, ed., Oligonucleotide Synthesis. A Practical Approach, (IRL Press, Washington, DC, 1984) or through the procedure of Example 25A.

J. 2'-Deoxy-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)cytidine-3'-O-(N,N-diisopropyl-β-cyanoethyl phosphoramidite).

2'-Deoxy-2'-fluorouridine is converted to the corresponding cytidine analog. The 5'-O-(4,4'-dimethoxy-trityl)-3'-O-(N,N-diisopropyl-β-cyanoethylphosphoramidite) can be prepared in accordance with Example 25A.

K. 2'-Deoxy-2'-fluoro-5'-O-(4,4'-dimethoxytrityl)guanosine-3'-O-(N,N-diisopropyl-β-cyanoethyl phosphoramidite).

2'-Deoxy-2'-fluoro guanosine was prepared according to the procedure of M. Ikehara and J. Imura, Chemical and Pharmaceutical Bulletin 29:1034-1038 and 3281-3285 (1981). This material was converted to the 5'-DMT-3'-phosphoramidite as described in Example 25A.

L. Preparation of 2'-Deoxy-2'-fluoro Modified Phosphorothioates Oligonucleotides.

2'-Deoxy-2'-substituted 5'-DMT nucleoside 3'-phosphoramidites prepared as described in Example 25 were inserted into sequence-specific oligonucleotide phosphorothioates as described by S. Beaucage et al., Journal of American Chemical Society 112:1253-1255 (1990) and B. S. Sproat, et al., Nucleic Acids Research 17:3373-3386 (1989).

    __________________________________________________________________________     SEQUENCE LISTING     (1) GENERAL INFORMATION:     (iii) NUMBER OF SEQUENCES: 121     (2) INFORMATION FOR SEQ ID NO: 1:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:     CCCCAACCACCTCTTGCTCC20     (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:     GTTCTCGCTGGTGAGTTTCA20     (2) INFORMATION FOR SEQ ID NO: 3:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:     AAAACGTCAGCCATGGTCCC20     (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:     GGATTCACTTCCACTGCGGG20     (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:     GAGACCCTGAACAGTTGATC20     (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:     CCCGGGAAAACGTCAGCCAT20     (2) INFORMATION FOR SEQ ID NO: 7:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:     CTGCCTCAGCGCCCCTTTGC20     (2) INFORMATION FOR SEQ ID NO: 8:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:     AGTCGGTGCAGTGGCTGGAG20     (2) INFORMATION FOR SEQ ID NO: 9:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:     GCAGAGGCTGGGGACATTGA20     (2) INFORMATION FOR SEQ ID NO: 10:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:     GGGCTGGGGAGGTGTTTGTT20     (2) INFORMATION FOR SEQ ID NO: 11:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:     CACTGCGGGGAGGGCTGGGG20     (2) INFORMATION FOR SEQ ID NO: 12:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:     AGCCGTGGCCTTAAAATTTT20     (2) INFORMATION FOR SEQ ID NO: 13:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:     ATTTTCAGGCCTCCATATGG20     (2) INFORMATION FOR SEQ ID NO: 14:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:     AAGAGAGAGACCCTGAACAG20     (2) INFORMATION FOR SEQ ID NO: 15:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:     GATAATGTTCTTGGTTGTAA20     (2) INFORMATION FOR SEQ ID NO: 16:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:     ATGGGGTGCACAAACTGGGG20     (2) INFORMATION FOR SEQ ID NO: 17:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:     GTCAGCCATGGTCCCCCCCC20     (2) INFORMATION FOR SEQ ID NO: 18:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:     CGCCGTGGAGTCGTTGCCCG20     (2) INFORMATION FOR SEQ ID NO: 19:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19:     TCAAATGGAGGCTGCCCGGC20     (2) INFORMATION FOR SEQ ID NO: 20:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20:     TGGAATCAGACACAAGCCGT20     (2) INFORMATION FOR SEQ ID NO: 21:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21:     CATCTTGCGCGCGGGGAGCC20     (2) INFORMATION FOR SEQ ID NO: 22:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:     TGCGCGCGGGGAGCCGGAGC20     (2) INFORMATION FOR SEQ ID NO: 23:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:     CGAGAGGTGCCGGCCCCGGG20     (2) INFORMATION FOR SEQ ID NO: 24:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:     CTCTCCTCGCCCTCCGTCGG20     (2) INFORMATION FOR SEQ ID NO: 25:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:     TGGAGTTTGCATTCACCTAC20     (2) INFORMATION FOR SEQ ID NO: 26:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:     AAAGGCCTCTAAGACAAGCT20     (2) INFORMATION FOR SEQ ID NO: 27:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 27:     GCCAGCATGTGCACCGTGAA20     (2) INFORMATION FOR SEQ ID NO: 28:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:     ACACCCCAGGCTCAACGATG20     (2) INFORMATION FOR SEQ ID NO: 29:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:     CCGAAGCTTACTCACAATTT20     (2) INFORMATION FOR SEQ ID NO: 30:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:     ACTTAGCTCTTGACTTCGGG20     (2) INFORMATION FOR SEQ ID NO: 31:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:     ATGCTGCGGAAAATAAATTG20     (2) INFORMATION FOR SEQ ID NO: 32:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:     ATTTTATTTTGAGCATGTTC20     (2) INFORMATION FOR SEQ ID NO: 33:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33:     TTTGGGGATGAGGGTGAGCA20     (2) INFORMATION FOR SEQ ID NO: 34:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:     CCCATTCCCACAGGCCTGAG20     (2) INFORMATION FOR SEQ ID NO: 35:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35:     CGGAGCGCGCCAGGCAGGGA20     (2) INFORMATION FOR SEQ ID NO: 36:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:     CCTTTTCCCAGACCAGCCAT20     (2) INFORMATION FOR SEQ ID NO: 37:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:     GGCCCCAGAAACGTAGCAGG20     (2) INFORMATION FOR SEQ ID NO: 38:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38:     GGATCCTGCCTTTCTTGGGG20     (2) INFORMATION FOR SEQ ID NO: 39:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39:     CAGCCATGGCCCCAGAAACG20     (2) INFORMATION FOR SEQ ID NO: 40:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:     CGACATGCCGGCGCCGCTGC20     (2) INFORMATION FOR SEQ ID NO: 41:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:     CAGACGACATGCCGGCGCCG20     (2) INFORMATION FOR SEQ ID NO: 42:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:     GCCTGCTTCGCAGCGGGAGA20     (2) INFORMATION FOR SEQ ID NO: 43:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43:     ACAGGTGCAGGAGTCGAGGC20     (2) INFORMATION FOR SEQ ID NO: 44:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:     GTCCCGTCTCAGGCCAGCCC20     (2) INFORMATION FOR SEQ ID NO: 45:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:     CCTCACCGATGCGGACCCTC20     (2) INFORMATION FOR SEQ ID NO: 46:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:     ATTGAACTTCATGGTGCCAG20     (2) INFORMATION FOR SEQ ID NO: 47:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:     TCTCACTCCCCATAAGGCTA20     (2) INFORMATION FOR SEQ ID NO: 48:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48:     TTCCTTTGGGTTCTCGTGCC20     (2) INFORMATION FOR SEQ ID NO: 49:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:     TTCCATCCTTCGACAGAGTT20     (2) INFORMATION FOR SEQ ID NO: 50:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:     AGGCTGATGCTGGGAAGGTC20     (2) INFORMATION FOR SEQ ID NO: 51:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:     GTTCTAAGGCTGATGCTGGG20     (2) INFORMATION FOR SEQ ID NO: 52:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 18     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:     TTCTCGCTGGTGAGTTTC18     (2) INFORMATION FOR SEQ ID NO: 53:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 17     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:     TCTCGCTGGTGAGTTTC17     (2) INFORMATION FOR SEQ ID NO: 54:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54:     AACTCGAGGTGGCCGCCGTC20     (2) INFORMATION FOR SEQ ID NO: 55:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:     CGCCTTCGCATAGCCCTTTG20     (2) INFORMATION FOR SEQ ID NO: 56:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:     GGAAGGGGTGATTGCGGGCC20     (2) INFORMATION FOR SEQ ID NO: 57:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:     AACACGCCCATTGCCCACCA20     (2) INFORMATION FOR SEQ ID NO: 58:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:     GTCTCAAGATGGCGTGCTCG20     (2) INFORMATION FOR SEQ ID NO: 59:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:     GCGATGGTTCAGCTGGGCCC20     (2) INFORMATION FOR SEQ ID NO: 60:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:     GCCCTCTCTCTCACTCCCCA2     (2) INFORMATION FOR SEQ ID NO: 61:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 61:     CTGGGAAGGTCCGATAGAGG20     (2) INFORMATION FOR SEQ ID NO: 62:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:     AAGGCTGATGCTGGGAAGGT20     (2) INFORMATION FOR SEQ ID NO: 63:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63:     GCAGGAACGGCGCCATGGTG20     (2) INFORMATION FOR SEQ ID NO: 64:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:     CTGGTTCGCCTCGTCCTCGG20     (2) INFORMATION FOR SEQ ID NO: 65:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:     ATCTGGATGACGCGCCCCTC20     (2) INFORMATION FOR SEQ ID NO: 66:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 66:     TTCTTGCAGCGCTCGGCCAG20     (2) INFORMATION FOR SEQ ID NO: 67:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 67:     TGCAATCCACGTCCTCCAGG20     (2) INFORMATION FOR SEQ ID NO: 68:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 68:     GGCTCCGCGGCGGTTCATCG20     (2) INFORMATION FOR SEQ ID NO: 69:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 69:     AAGCGGTGCGGCATGTCGAT20     (2) INFORMATION FOR SEQ ID NO: 70:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 70:     GCAGGCTGCCGCAGTGGTCA20     (2) INFORMATION FOR SEQ ID NO: 71:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 71:     CCTCCCCAGCAACTCCGGTC20     (2) INFORMATION FOR SEQ ID NO: 72:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 72:     AGCGGCCTTTGTCCTGGATG20     (2) INFORMATION FOR SEQ ID NO: 73:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 73:     GGCCATCCCGGTCCAACAGC20     (2) INFORMATION FOR SEQ ID NO: 74:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 74:     GGTGCTGGCCCGGCTCTCCC20     (2) INFORMATION FOR SEQ ID NO: 75:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 75:     GGACCCCGAAAGACCACCAG20     (2) INFORMATION FOR SEQ ID NO: 76:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 76:     GTGGCTCCAACCTCCGCTTT20     (2) INFORMATION FOR SEQ ID NO: 77:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 77:     AGGAGGTGCTCGAATTTGGG20     (2) INFORMATION FOR SEQ ID NO: 78:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 78:     ACTACCATGGTCGGGGCGGG20     (2) INFORMATION FOR SEQ ID NO: 79:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 79:     GTCCCACCGCATGGCGCAGC20     (2) INFORMATION FOR SEQ ID NO: 80:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 80:     GTTTGGCCGATGCGCGAGTC20     (2) INFORMATION FOR SEQ ID NO: 81:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 81:     TGCAGTTGGCCACGAAGTCG20     (2) INFORMATION FOR SEQ ID NO: 82:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 82:     GTGGGGCATGTTGACGCTGA20     (2) INFORMATION FOR SEQ ID NO: 83:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 83:     CCAGAGCAGGGACCCACAGT20     (2) INFORMATION FOR SEQ ID NO: 84:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 84:     TCTCCTCGGTTGTCAAATGA20     (2) INFORMATION FOR SEQ ID NO: 85:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 85:     CGGTGCTCCTCTCCTCGGTT20     (2) INFORMATION FOR SEQ ID NO: 86:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 86:     AGCCAAAATCCTCTTCTCTG20     (2) INFORMATION FOR SEQ ID NO: 87:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 87:     CATGAGGGCCGATGTGACCT20     (2) INFORMATION FOR SEQ ID NO: 88:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 88:     ATCCCTTCCTTGCACATCCC20     (2) INFORMATION FOR SEQ ID NO: 89:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 89:     CCCCAGGGCCCACCAGTCCA20     (2) INFORMATION FOR SEQ ID NO: 90:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 90:     AGCACCCCCAGGGCCCACCA20     (2) INFORMATION FOR SEQ ID NO: 91:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 91:     CGTACATCAGCACCCCCAGG20     (2) INFORMATION FOR SEQ ID NO: 92:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 92:     CCAGCCATCATCTCGTACAT20     (2) INFORMATION FOR SEQ ID NO: 93:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 93:     TGCCACACAGCCCAGGCGCA20     (2) INFORMATION FOR SEQ ID NO: 94:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 94:     TCAGGGCATCAGGTCTTCAC20     (2) INFORMATION FOR SEQ ID NO: 95:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 95:     TCTCAGGGCATCAGGTCTT20     (2) INFORMATION FOR SEQ ID NO: 96:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 96:     AAGGAAAGTCTGCGGCCGGG20     (2) INFORMATION FOR SEQ ID NO: 97:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 97:     TGGCGGCTCCCGTTCTGCAG20     (2) INFORMATION FOR SEQ ID NO: 98:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 98:     GCTTCCTCGGCCGCATGCGT20     (2) INFORMATION FOR SEQ ID NO: 99:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 99:     TTGACGCTGAACCGCTGGGA20     (2) INFORMATION FOR SEQ ID NO: 100:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 100:     GCCCGGTGCTCCTCTCCTCG20     (2) INFORMATION FOR SEQ ID NO: 101:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 101:     GGGCCGATGTGACCTCTGCA20     (2) INFORMATION FOR SEQ ID NO: 102:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 102:     TGGAGGAACATGAGGGCCGA20     (2) INFORMATION FOR SEQ ID NO: 103:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 103:     CCCCCAGGGCCCACCAGTCC20     (2) INFORMATION FOR SEQ ID NO: 104:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 104:     TGCGATGCCACACAGCCCAG20     (2) INFORMATION FOR SEQ ID NO: 105:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 105:     TGGGCTCTCAGGGCATCAGG20     (2) INFORMATION FOR SEQ ID NO: 106:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 106:     CGCCGCTCCCTTCCATCTTG20     (2) INFORMATION FOR SEQ ID NO: 107:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 107:     CCCCGTAATGCGCCTTGAGG20     (2) INFORMATION FOR SEQ ID NO: 108:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 108:     CTGTCCACCCACTTGAGGGT20     (2) INFORMATION FOR SEQ ID NO: 109:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 109:     TTGGAAGAGGTGGCCGTTGG20     (2) INFORMATION FOR SEQ ID NO: 110:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 110:     CCTGTTAAAGCGCTTGGCTT20     (2) INFORMATION FOR SEQ ID NO: 111:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 111:     TGCAGGTCAGCGGGACGAGG20     (2) INFORMATION FOR SEQ ID NO: 112:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 112:     AGCCCCTGAGAGATTTTGAT20     (2) INFORMATION FOR SEQ ID NO: 113:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 113:     TTCTTCAACCGCACCAGGAG20     (2) INFORMATION FOR SEQ ID NO: 114:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 114:     TCCTTGCACATGCCGTAGTC20     (2) INFORMATION FOR SEQ ID NO: 115:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 115:     GGAGCGCCCGGCCATCATCT20     (2) INFORMATION FOR SEQ ID NO: 116:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 116:     GGGCTCGCTGGTGAACTGTG20     (2) INFORMATION FOR SEQ ID NO: 117:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 117:     GACGCACGCGGCCTCACACC20     (2) INFORMATION FOR SEQ ID NO: 118:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 118:     TCGGAGCCGTGCCCAGCCTG20     (2) INFORMATION FOR SEQ ID NO: 119:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 119:     CGGGCCAGGTGTGAGGGACT20     (2) INFORMATION FOR SEQ ID NO: 120:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 120:     CCGCGACGCAGGCACAGCAG20     (2) INFORMATION FOR SEQ ID NO: 121:     (i) SEQUENCE CHARACTERISTICS:     (A) LENGTH: 20     (B) TYPE: nucleic acid     (C) STRANDEDNESS: single     (D) TOPOLOGY: linear     (iv) ANTI-SENSE: yes     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 121:     GGTCAGTGCATCGAGTTCTG20     __________________________________________________________________________ 

What is claimed is:
 1. An antisense oligonucleotide up to 50 nucleotide units in length comprising SEQ ID NO: 2, which oligonucleotide specifically binds human protein kinase C-α mRNA.
 2. The oligonucleotide of claim 1 comprising at least one phosphorothioate linkage.
 3. The oligonucleotide of claim 1 comprising at least one 2'-O-alkyl or 2'-fluoro modification.
 4. A composition comprising the oligonucleotide of claim 1 and a pharmaceutically acceptable carrier or diluent.
 5. A composition comprising the oligonucleotide of claim 1 and a chemotherapeutic agent.
 6. A method of inhibiting human protein kinase C-α expression in cells comprising contacting the cells with an oligonucleotide of claim
 1. 7. The method of claim 6 wherein the cells are cancer cells. 